Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch

Donald W. Lawrence, Katrina M. Comerford, and Sean P. Colgan

Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.

barrier function; restitution; protein kinase A; vasodilator-stimulated phosphoprotein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPITHELIAL CELLS OF MANY ORGANS function as a barrier for maintaining a physical and selective interface between external and internal environments. The regulation of epithelial permeability is strictly controlled. Tight junctions, along with adherens junctions, form the apical junctional complex (45). Barrier function must be maintained to restrict the movement of potentially harmful and immunogenic materials across the epithelial cell layer while allowing diffusion of ions and solutes. The tight junction also restricts the movement of lipids and integral membrane proteins between the apical and basolateral domains (60).

Convincing evidence indicates that the actin cytoskeleton substantially contributes to dynamic changes in epithelial and endothelial permeability (38, 61). Disruption of the microfilament cytoskeleton with the phallotoxin cytochalasin B decreases barrier function in Caco-2 cells (5), while F-actin-stabilizing agents, such as phalloidin, increase barrier function (62). Moreover, inactivation of the small GTPase rho with a chimeric Clostridium difficile toxin, DC3B, is associated with the disappearance of the apical perijunctional actin ring and increased paracellular permeability (50). Current hypotheses suggest that epithelial paracellular permeability is regulated by circumferential contraction of the perijunctional actin ring (41).

In light of the relationship between paracellular permeability and the perijunctional actin ring, understanding spatially confined actin polymerization dynamics is key to elucidating a precise mechanism for regulation of barrier function by the cytoskeleton. The vasodilator-stimulated phosphoprotein (VASP) is of particular interest given its actin-binding and actin monomer-nucleating capabilities. VASP was originally discovered as a focal adhesion protein, targeted to areas of the cell that undergo dynamic actin polymerization (i.e., lamellapodia) (53). Reports that demonstrated VASP binding to profilin and ActA, a surface protein of Listeria monocytogenes, provided evidence that VASP may play a role in assembly of the actin microfilament network (11). VASP is composed of three major domains: an EVH1 domain, an EVH2 domain, and a central proline-rich region (22). The EVH1 domain binds to proline-rich (E/D)FPPPPX(D/E) motifs (48) present in VASP binding proteins. Actin-binding and cross-linking capabilities are conferred via the COOH-terminal EVH2 domain (3). VASP is a known substrate for protein kinase A (PKA, cAMP dependent) and protein kinase G (PKG, cGMP dependent) (24, 59), and three phosphorylation sites are shared by PKA and PKG: S157, S239, and T278. Phosphorylation at Ser157 is the preferred PKA phosphorylation site and results in a conformational change in VASP and a shift in SDS-PAGE mobility from 46 to 50 kDa (30). The function of this conformational change is not known. Recently, Harbeck et al. (25) demonstrated that VASP phosphorylation by PKA does not influence the binding of zyxin, vinculin, or profilin but significantly diminishes VASP-actin association. Such studies provide the basis to examine the role of VASP in regulation of tight junction permeability. Here, we investigate the involvement of cAMP signal transduction in regulating epithelial permeability during tight junction assembly. Biochemical, morphological, and functional data provide strong evidence that VASP may provide a critical link in tight junction regulatory processes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. T84 cells, a columnar intestinal crypt epithelial cell line, were used as a model low-permeability cell line. When plated on permeable supports, T84 cells form high-resistance monolayers and have been used to study a number of aspects of epithelial barrier function. KB cells are squamous cervical epithelial cells and were used as a high-permeability cell type for comparison. Epithelial cells were grown on permeable 0.33-cm2 ring-supported polycarbonate filters (0.4 µm pore size; Costar, Cambridge, MA) or plastic polystyrene tissue culture dishes (Costar) using previously described techniques (14, 42).

Ca2+ switch experiments. Epithelial cells were grown to electrical confluence [transepithelial resistance (TER) > 1,000 ohm · cm2] on permeable inserts and used 7-10 days after they were plated. For Ca2+ switch experiments, extracellular Ca2+ was chelated with 2 mM EDTA for 5 min at 37°C, and TER was monitored to ensure that TER had maximally fallen, as described previously (52). Monolayers were then washed in Hanks' balanced salt solution (HBSS), and TER was monitored using probes interfaced with a voltmeter (Evohm, World Precision Instruments, New Haven, CT) or a voltage clamp (Iowa Dual Voltage Clamps, Dept. of Bioengineering, University of Iowa) interfaced with an equilibrated pair of calomel and Ag-AgCl electrodes, as described in detail elsewhere (39). Where indicated, PKA was inhibited by treatment with 3-30 µM H-89 (Sigma, St. Louis, MO) for 60 min at 37°C before Ca2+ switch or activated by treatment with 0.1-10 µM 5'-N-ethylcarboxamidoadenosine (NECA; Calbiochem, La Jolla, CA) for 10 min at 37°C before Ca2+ switch. Monolayer recovery was allowed to proceed in the presence of NECA or H-89.

Immunoprecipitation and immunoblotting experiments. For immunoprecipitation experiments, T84 or KB cells were grown to confluence on 100-mm plastic petri dishes. Monolayers were lysed for 10 min in 1 ml of low-stringency lysis buffer (150 mM NaCl, 25 mM Tris, pH 8.0, 5 mM EDTA, 0.25% Triton X-100, 0.25% Igepal, and 10% mammalian tissue protease inhibitor cocktail; Sigma) to preserve protein-protein interactions. For immunoblotting of VASP, monolayers were lysed in the above-described lysis buffer containing 1% Triton X-100 and 1% Igepal. Monolayers were scraped, and the lysates were collected in microfuge tubes. After lysates were spun at 14,000 g to remove cell debris, the pellet was discarded. For immunoprecipitations, lysates were precleared with 50 µl of a 50% protein G-Sepharose slurry (Amersham Pharmacia, Piscataway, NJ) for 2 h at 4°C. Anti-VASP or anti-ZO-1 (2.5 µg) was added to 1 ml of lysate, rotated overnight at 4°C, and then subjected to capture with 100 µl of 50% protein G-Sepharose slurry. The captured antigen was washed three times in lysis buffer to avoid disruption of protein-protein complexes. Proteins were solubilized in Laemmli sample buffer and heated to 100°C for 5 min. Samples were resolved on a 7.5% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in PBS supplemented with 0.2% Tween 20 (PBS-T) and 4% BSA. Membranes were incubated with monoclonal anti-VASP (125 ng/ml; Transduction Laboratories, Lexington, KY) in PBS-T for 1 h at room temperature and washed for 3-10 min in PBS-T. Membranes were then incubated with peroxidase-conjugated goat-anti mouse IgG (110 ng/ml; ICN/Cappel, Costa Mesa, CA) for 1 h at room temperature. After the wash was repeated, proteins were detected by enhanced chemiluminescence.

Generation of anti-phospho-VASP antibody. Anti-phospho-VASP antibody was generated by immunizing rabbits with the following serine-phosphorylated peptide coupled to keyhole limpet hemocyanin: EHIERRVS(PO3)NAGGPPA. This peptide antigen represents the PKA phosphorylation site (Ser157 flanked by 7 amino acids on each side) within VASP that induces the characteristic conformational change on phosphorylation. To purify the phosphospecific antibody, the rabbit serum was passed over a column of cyanogen bromide-activated Sepharose 4B coupled to the phosphopeptide immunogen. The column was washed repeatedly with wash buffer (50 mM Tris, pH 8.0, 150 mM NaCl). The bound phosphoserine-specific antibodies were eluted in 1-ml fractions with 100 mM glycine and neutralized immediately with 1 M Tris (pH 8.0). The eluant was combined and passed over a column containing unphosphorylated peptide to remove antibodies that recognize nonphosphorylated VASP. Flow through contained antibodies specific for VASP protein phosphorylated at Ser157.

Immunofluorescent staining of epithelial monolayers. KB or T84 cells were grown to confluence on acid-washed 12-mm glass coverslips. Where indicated, monolayers were treated with 30 to 0.3 µM H-89 for 60 min before the addition of 10 µM NECA for 10 min at 37°C. Monolayers were washed once in PBS and fixed for 10 min at room temperature in 1% paraformaldehyde in cacodylate buffer (0.1 M sodium cacodylate, pH 7.4, 0.72% sucrose). The monolayers were permeabilized for 10 min in PBS containing 0.2% Triton X-100 and 3% BSA. After they were washed twice with PBS, the cells were stained for 1 h with a monoclonal anti-VASP (12.5 µg/ml) antibody, a polyclonal anti-phospho-VASP antibody (11 µg/ml), and/or a polyclonal antibody to ZO-1 (1 µg/ml; Zymed, San Francisco, CA). After they were washed, the monolayers were incubated with goat anti-mouse Oregon green (1 µg/ml) or goat anti-mouse Texas red (1 µg/ml). Fluorescently labeled secondary antibodies were purchased from Molecular Probes (Eugene, OR). Laser Sharp imaging software (Bio-Rad, Hercules, CA) was used to determine protein colocalization. Specific areas were determined by first generating a two-dimensional colocalization fluorogram using the red and green intensities of the combined image and then selectively displaying only those pixels that are colocalized (yellow).

Plasmids and transfections. T84 cells were grown on permeable polycarbonate inserts and transfected 7-10 days after plating. Plasmids based on the pcDNA3 expression vector were kindly provided by Dr. Matthias Reinhard (Institute for Clinical Biochemistry and Pathobiochemistry, Medical University of Wurzburg, Wurzburg, Germany) (3). The plasmids were transiently transfected in T84 cell monolayers using the GenePorter transfection system (Gene Therapy Systems, San Diego, CA). Monolayers were used in Ca2+ switch experiments 2 days after transfection. To confirm successful transfection of truncated VASP, monolayers were solubilized in reducing SDS sample buffer and subjected to SDS-PAGE and Western blot analysis for VASP. The presence of relevant VASP truncations by Western blot analysis confirmed expression.

Data presentation. Electrophysiological data were compared by two-factor analysis of variance (ANOVA) or by Student's t-test where appropriate. Values are means ± SE of n monolayers from at least three separate experiments.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of PKA in barrier resealing during Ca2+ switch. Chelation of extracellular Ca2+ induces tight junction disassembly in epithelial (32) and endothelial cells (26). The return to normal Ca2+ concentration (i.e., Ca2+ switch) initiates tight junction reassembly and recovery of normal barrier function. Moreover, elevation in intracellular cAMP levels has been shown to promote epithelial barrier function after disruption, such as occurs with polymorphonuclear (PMN) transmigration (17). The signaling mechanisms for such enhanced junctional assembly are not completely understood. To investigate the contribution of cAMP to tight junction assembly, we examined epithelial barrier recovery after Ca2+ switch in the presence and absence of the adenosine receptor agonist NECA, a potent activator of epithelial adenylyl cyclase via the adenosine A2 receptor (6). Consistent with previous studies (17), incubation of epithelia with NECA alone (10 µM) did not induce a significant change in TER in fully confluent T84 cell monolayers (106 ± 8 vs. 100 ± 3% of baseline TER for HBSS- and NECA-treated monolayers, respectively, not significant; Fig. 1A). In monolayers preexposed to 2 mM EDTA for 5 min and then returned to tissue culture medium containing normal Ca2+ levels for 3 h, TER values recovered to 49 ± 4% of baseline TER. Treatment with increasing concentrations of NECA resulted in a concentration-dependent increase in barrier recovery after Ca2+ switch (P < 0.01 by ANOVA), with maximally stimulated recovery at 1 µM NECA (93 ± 3% of baseline TER, P < 0.025 compared with EDTA treatment). Such data suggest that agents that elevate cAMP enhance tight junction reassembly.


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Fig. 1.   cAMP signal transduction pathway is involved in epithelial barrier recovery after Ca2+ switch. A: Ca2+ switch experiments were performed on confluent T84 monolayers in the presence and absence of various concentrations of the adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA). NECA had no significant influence on transepithelial resistance (TER) in control T84 monolayers. However, NECA at all concentrations tested significantly enhanced barrier recovery 180 min after Ca2+ switch (P = 0.003 by ANOVA). HBSS, Hanks' balanced salt solution. B: T84 monolayers were treated with indicated concentrations of H-89 to inhibit protein kinase A (PKA). Inhibition of PKA with H-89 significantly attenuated T84 barrier recovery 180 min after Ca2+ switch (P < 0.001 by ANOVA). Barrier recovery was significantly diminished in the presence of 10 µM H-89 (P = 0.03) and 30 µM H-89 (P = 0.001). Treatment with 30 µM H-89 induced a 60% decrease in barrier recovery. Values are means ± SE of 3 independent experiments.

Intracellular elevation of cAMP activates PKA; therefore, we next examined the role of PKA in epithelial barrier recovery after Ca2+ switch. To do this, a PKA inhibitor approach was adopted. As shown in Fig. 1B, treatment of confluent T84 monolayers with H-89, a specific PKA inhibitor (20), attenuated barrier recovery in a concentration-dependent fashion (P < 0.01 by ANOVA). Monolayers treated with H-89 alone (30 µM) showed a slightly significant attenuation of barrier recovery vs. control monolayers (P = 0.04). Taken together, these data indicate that cAMP signal transduction mechanisms are likely to be a significant part of the regulatory pathways that govern tight junction reassembly during epithelial resealing.

PKA-dependent VASP phosphorylation in epithelial cells. The architecture of the actin cytoskeleton is crucial for tight junction biogenesis and maintenance (13). Critical to tight junction integrity is the circumferential cortical actin ring located at the level of the tight junction in polarized cells (50, 57). On the basis of the involvement of cAMP-mediated signal transduction in barrier recovery and recent work indicating that VASP may localize to epithelial cell-cell junctions (58), we next investigated the role of VASP, a PKA effector protein that modulates actin cytoskeletal dynamics (35). As shown in Fig. 2A, epithelial exposure to 10 µM NECA resulted in rapid VASP phosphorylation (shift from 46 to 50 kDa). Indeed, on exposure, VASP was rapidly phosphorylated (within 30 s), with sustained phosphorylation beyond 5 min. Longer exposure (>30 min) also revealed detectable VASP phosphorylation under these conditions (data not shown). Forskolin (10 µM, 5 min at 37°C), an activator of adenylyl cyclase, was used as a positive control and also induced VASP phosphorylation. VASP is a known substrate for PKA and PKG; therefore, we used the specific PKA inhibitor H-89 to dissect the role of PKA in NECA- and forskolin-induced VASP phosphorylation (Fig. 2B). Treatment with H-89 induced a concentration-dependent decrease in VASP phosphorylation. Such data confirm PKA-dependent VASP phosphorylation under conditions where intracellular cAMP levels are elevated. We also examined the amounts of VASP and phospho-VASP in the pellet fraction of our lysates. These experiments revealed very little VASP or phospho-VASP under our lysis conditions (data not shown). Our choice of lysis buffer (containing Triton X-100 and Igepal) may not be best for studying VASP-actin interactions, and thus it is difficult to draw strong conclusions from these experiments. Moreover, in light of the published fact that VASP may not strongly associate with actin under conditions in which it is phosphorylated (25), the soluble fraction may be the more relevant fraction.


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Fig. 2.   Vasodilator-stimulated phosphoprotein (VASP) phosphorylation in intestinal epithelial cells. A: VASP was predominantly expressed as an unphosphorylated protein [control (C), lane 1]. P-VASP, phospho-VASP. Forskolin (Fsk, 10 µM) was used as a positive control (lane 2). Stimulation with 10 µM NECA induced VASP phosphorylation from 30 s to 5 min (lanes 3-6). B: inhibition of VASP phosphorylation by H-89. T84 monolayers were treated with or without various concentrations of H-89 in the presence and absence of 10 µM NECA or 10 µM forskolin. NECA and forskolin induced VASP phosphorylation over control levels. Treatment with 30 µM H-89 alone (H-89) minimally inhibited constitutive VASP phosphorylation. Forskolin- and NECA-induced VASP phosphorylation was reduced in the presence of the PKA inhibitor H-89. Maximal inhibition was observed with 30 µM H-89.

VASP is phosphorylated during epithelial barrier recovery. Use of the Ca2+ switch model allows the opportunity to investigate functional signaling mechanisms during rebuilding of the tight junction. Thus we correlated VASP phosphorylation with barrier recovery during Ca2+ switch (Fig. 3). Brief treatment with 2 mM EDTA induced a decrease in TER to ~20% of baseline values. Within 3 h, resistance recovered to ~60% of baseline values. The largest increase in TER occurred within the 1st h after return to normal Ca2+ levels (Fig. 3A). Monolayers were harvested at various time points and assayed by Western blot to assess the state of VASP phosphorylation (Fig. 3B). Consistent with previous studies, some constitutive VASP phosphorylation was evident (23), and exposure to 2 mM EDTA did not significantly alter basal phosphorylation. Conversely, within the first 60 min of resealing, a nearly eightfold increase in phospho-VASP was evident (densitometric analysis; Fig. 3C), and this correlated with the greatest rate of monolayer resealing (5-60 min, slope = 0.44% change/min; Fig. 3A). After 1 h, we observed a rapid loss of phospho-VASP that was correlated with a significant decrease in the rate of monolayer resealing (60-180 min, slope = 0.11% change/min), representing an ~80% decrease in the rate of monolayer resealing. These data implicate a role for VASP in epithelial resealing after Ca2+ switch.


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Fig. 3.   VASP phosphorylation during epithelial barrier recovery. A: confluent T84 monolayers were treated with 2 mM EDTA for 5 min and then incubated in cell culture medium with normal Ca2+ levels for 3 h. Return to normal-Ca2+ medium reestablishes barrier integrity over time. B: sample monolayers were taken at various time points during a Ca2+ switch experiment to determine phosphorylation state of VASP. Treatment with EDTA did not significantly influence VASP phosphorylation. VASP phosphorylation increased ~8-fold over that of EDTA-treated monolayers during the 1st h of resealing and decreased below baseline levels. Unt, untreated. C: densitometric analysis of VASP phosphorylation during epithelial resealing (representative of 3 experiments).

VASP localization in epithelial cells. Confocal laser scanning microscopy was utilized to determine the subcellular localization of VASP in confluent high-resistance (T84 cells, TER > 1,000 Omega  · cm2) and low-resistance (KB cells, TER < 50 Omega  · cm2) epithelia. Staining for the tight junction marker ZO-1 revealed intense staining in a classic "chicken-wire" pattern (Fig. 4A). In polarized, high-resistance T84 cells, VASP was localized subapically in a pattern indicative of tight junction association. Specific areas of VASP-ZO-1 colocalization were demonstrated by computer-generated overlay, and imaging of VASP and ZO-1 in transverse x-z image (Fig. 4B) revealed distinct colocalization as well as areas of only ZO-1 staining. VASP is a focal adhesion protein and binds to other known focal adhesion proteins such as zyxin and vinculin (29). In T84 cells, VASP was also localized to focal adhesions at the terminal ends of basally located stress fibers (data not shown). In low-resistance epithelial cells (KB cells; Fig. 4C), the classic chicken-wire ZO-1 staining pattern common to high-resistance T84 monolayers was not evident, and the staining pattern was considerably more diffuse at areas of cell-cell contact. In KB monolayers, VASP stained intensely at areas of cell-cell contact, and ZO-1-VASP colocalization was evident. Such data provide morphological evidence for VASP localization at the epithelial tight junction and strengthen the hypothesis that VASP may be a contributing factor in tight junction assembly after Ca2+ switch.


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Fig. 4.   VASP is associated with ZO-1 at epithelial tight junctions. Confluent T84 or KB monolayers were stained for VASP, ZO-1, or VASP and ZO-1 simultaneously to determine whether VASP is localized to the region of the tight junction. Stained monolayers were analyzed at the level of the tight junction by confocal laser scanning microscopy. A: in T84 cells, staining at the level of the tight junction for ZO-1 was observed as the characteristic "chicken-wire" staining pattern. Staining for VASP resulted in a nearly identical staining pattern. Indeed, a computer-generated image of specific colocalized areas (yellow) supports VASP localization to the tight junction. B: transverse x-z image also demonstrates VASP-ZO-1 colocalization to the tight junction complex. C: in KB cells, intense ZO-1 and VASP staining was colocalized only in areas of cell-cell contact. D: VASP or ZO-1 immunoprecipitates (IP) from T84 monolayers were cross-blotted for ZO-1 or VASP, respectively. Lane 1, IP reaction using an irrelevant control antibody; lane 2, IP reaction with specific antibody; lane 3, T84 lysate run as a positive standard. The presence of VASP in a ZO-1 IP and ZO-1 in a VASP IP suggests that VASP may be a novel member of the tight junction complex. WB, Western blot.

To support our finding that VASP and ZO-1 are associated in high- and low-resistance cells, a biochemical approach was taken (Fig. 4D). Confluent T84 and KB monolayers were lysed in low-stringency lysis buffer to preserve protein-protein interactions. Lysate from T84 cells was immunoprecipitated with anti-VASP or anti-ZO-1, and resulting Western blots were immunoblotted with anti-ZO-1 or anti-VASP, respectively. As shown in Fig. 4D, ZO-1 was present in immunoprecipitation reactions specific for VASP; likewise, VASP was present in immunoprecipitations for ZO-1, thus confirming the morphological findings that VASP is associated with the tight junction marker ZO-1. On the basis of these studies, it was uncertain how much VASP is associated with ZO-1 at the tight junction. In addition, these findings do not necessarily indicate a direct association between ZO-1 and VASP, only that they exist within the same tight junction complex.

We investigated the localization of phospho-VASP at the tight junction, an actin-rich structure, under conditions in which VASP would be phosphorylated predominantly by PKA. To do this, we generated a phosphospecific VASP anti-peptide antibody using a Ser157-phosphorylated peptide corresponding to residues 149-164 as an antigen. To investigate the localization of phospho-VASP during conditions in which cAMP levels are elevated, confluent T84 monolayers were treated with 10 µM NECA and immunolocalized for phospho-VASP (Fig. 5A). Little or no phospho-VASP was evident at the level of the tight junction in unstimulated T84 monolayers (Fig. 5Aa). A time course of NECA exposure revealed the rapid (1 min) appearance of phospho-VASP within the junction, and such staining was maximal by 10 min of exposure. Exposure for >20 min revealed a slow dissipation of staining within the area of the junction. As shown in Fig. 5B, treatment with the PKA inhibitor H-89 abolished phospho-VASP staining at the level of the tight junction in NECA-treated monolayers. Intestinal epithelial cells were treated with 10-fold dilutions of H-89 (30 to 0.3 µM) and then stimulated with 10 µM NECA. Phospho-VASP localization to the tight junction was completely abolished in monolayers treated with the maximal concentration of H-89 (Fig. 5Ba). Phospho-VASP localization was observed in monolayers treated with 3 or 0.3 µM H-89 (Fig. 5B, b and c, respectively). To examine the specificity of our anti-phospho-VASP antibody, T84 lysate treated with 10 µM NECA was immunoblotted for VASP, stripped, and reprobed for phospho-VASP (Fig. 5C). The phospho-VASP antibody recognized only the 50-kDa form of VASP, corresponding to Ser157 phosphorylation and the associated shift in relative molecular weight. To verify the necessity for phosphorylation recognition by this antibody, phospho-VASP immunoprecipitates were exposed to calf intestinal alkaline phosphatase (50 U, 2 h, 37°C) and examined for immunoreactivity by immunoblot. Pretreatment with alkaline phosphatase resulted in a >90% loss of recognition by the phospho-VASP antibody (data not shown). These data provide evidence that PKA activation results in the appearance of phospho-VASP at the level of the epithelial tight junction. It is not known, however, whether VASP phosphorylation results in the translocation of phospho-VASP to the tight junction or PKA phosphorylates VASP already present in the tight junction-protein complex.


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Fig. 5.   Phospho-VASP is localized to tight junctions in NECA-treated epithelial monolayers. A: T84 monolayers on glass coverslips were treated with 10 µM NECA for 1 min (b), 5 min (c), 10 min (d), 20 min (e), or 30 min (f) and then stained with anti-phospho-VASP. a, Unstimulated monolayers. Phospho-VASP is localized to the tight junction under conditions in which cAMP is elevated (arrows). B: confluent monolayers were treated with 30 µM (a), 3 µM (b), or 0.3 µM (c) H-89 and then stimulated with 10 µM NECA for 10 min. H-89 (30 µM) completely abolished the localization of phospho-VASP to the tight junction (a). Phospho-VASP at the tight junctions was observed in monolayers treated with 3 or 0.3 µM H-89 (b and c, respectively). C: to show specificity of the polyclonal anti-phospho-VASP antibody, platelet VASP lysate was immunoblotted using an antibody that recognizes both forms of VASP (VASP). Blot was stripped and reprobed with anti-phospho-VASP (P-VASP). A single 50-kDa band was observed, suggesting that the anti-phospho-VASP antibody is specific for phosphorylated VASP.

Localization of phospho-VASP in T84 monolayers after Ca2+ switch. Recent studies have demonstrated that Ca2+ switch results in the loss of a number of key regulatory proteins in the tight junction (13). Therefore, we investigated the localization of VASP and phospho-VASP in T84 monolayers after Ca2+ switch (Fig. 6). In untreated monolayers, phospho-VASP was diffuse with no evident structural localization, while VASP remained localized to the tight junction. Treatment with 2 mM EDTA did not change the immediate localization of VASP or phospho-VASP. However, as shown in Fig. 6, phospho-VASP immunolocalization during barrier recovery revealed the dominant appearance of phospho-VASP in the tight junction and colocalization with dephosphorylated VASP. These data provide morphological evidence that VASP phosphorylation may play a role in reestablishing barrier integrity after Ca2+ switch.


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Fig. 6.   Phospho-VASP is localized to the tight junction after Ca2+ switch. Confluent T84 monolayers on glass coverslips were incubated in 2 mM EDTA to disrupt tight junction integrity and then returned to normal-Ca2+ (NC) medium for 3 h. In untreated monolayers, VASP was localized to the tight junction, while phospho-VASP staining was diffuse and minimally localized to the tight junction. VASP (green) and phospho-VASP (red) were not colocalized in control monolayers. VASP localization to the tight junction was diminished in monolayers treated with EDTA. At 2 h after the Ca2+ switch, VASP and phospho-VASP were colocalized at the level of the tight junction. The most intense colocalization was found after 3 h in normal-Ca2+ medium.

Expression of COOH-terminal human VASP fragments diminishes barrier recovery after Ca2+ switch. To dissect the role of phospho-VASP in epithelial barrier recovery after Ca2+ switch, truncated forms of VASP were expressed in epithelial cells and functionally examined. To do this, plasmids expressing amino acids 259-342 within the EVH2 domain (Bl mutant) or amino acids 277-380 of the EVH2 domain (lC mutant) were transiently transfected in T84 cells. Previous studies have indicated three conserved regions of amino acids in the EVH2 domain of VASP (blocks A, B, and C) (3). Fragments lacking one or more of these conserved regions have altered actin-binding and oligomerization ability. The Bl fragment lacks the conserved block C and has diminished actin-binding capacity compared with wild-type VASP and will form only monomers and dimers under nonreduced conditions, while the lC fragment (lacking block B) will tetramerize normally but does not bind actin. Additionally, each of these truncations lacks the ability to be phosphorylated at Ser157. Interestingly, the expression of the truncated VASP did not alter baseline TER compared with mock-transfected monolayers (baseline TER = 1,085 ± 73, 1,108 ± 31, and 1,205 ± 50 Omega  · cm2 for Bl, lC, and mock-transfected fragments, respectively, not significant). However, as shown in Fig. 7, transient transfection of COOH-terminal VASP fragments significantly inhibited monolayer recovery during the 3-h reestablishment of barrier. As shown in Fig. 7B, these results are from cells expressing a significant amount of wild-type VASP protein. Confirmation of truncated VASP expression revealed the predicted 13- and 11-kDa proteins (arrows) as well as the 46-kDa wild-type protein (Fig. 7B). Such observations indicate that full-length VASP is necessary for reestablishment of epithelial barrier after Ca2+ switch. Taken together, these data identify VASP and structurally modified phospho-VASP as key components in the dynamic regulation of epithelial tight junctions. Transfection of T84 cells has been achieved (43, 44); however, highly efficient DNA incorporation has been somewhat problematic. Therefore, using densitometric analysis of the Western blot in Fig. 7B, we determined the amount of mutant VASP fragment that was expressed relative to the amount of total VASP. The Bl fragment comprised 17% of the total VASP, while the lC fragment made up 34% of the total VASP protein. The difference in expressed VASP mutant may actually be related to the actin-binding abilities of each fragment and not to a difference in the amount of expressed protein. The Bl fragment binds actin to a higher degree than the lC fragment (3); therefore, under our lysis conditions, a portion of the Bl fragment may be associated with the actin cytoskeleton and may be unintentionally discarded as part of the lysis procedure. Such an occurrence would be perceived as a decrease in expression, when in fact it is a matter of less protein available during the Western blotting procedure. Both fragments attenuated barrier recovery to about the same extent (Fig. 7A).


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Fig. 7.   Transient transfection of COOH-terminal VASP fragments alters monolayer ability to reseal after Ca2+ switch. T84 monolayers were transiently transfected with plasmids encoding COOH-terminal fragments of VASP (Bl, encoding amino acids 259-342, and lC, encoding amino acids 277-380) and then subjected to Ca2+ switch. A: TER was measured 3 h after Ca2+ switch. Expression of the COOH-terminal VASP fragments significantly diminished monolayer resealing. Values are means ± SE of 19 monolayers per condition. dagger  P < 0.001; * P < 0.05 vs. mock-transfected fragments (Mck). B: to confirm transfection, monolayers from each condition were immunoblotted for VASP. Arrows, low-molecular-weight COOH-terminal VASP fragments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In intact epithelia, tight junctions comprise a formidable barrier to macromolecules. Paracellular permeability is tightly controlled, in part by the tight junction complex; however, precise mechanisms involved in regulating tight junction assembly and control of paracellular permeability are not fully elucidated. Here, we demonstrate that VASP is implicated in PKA-mediated modulation of tight junction permeability.

A number of signal transduction pathways have been demonstrated to directly regulate tight junction assembly, including activation of protein kinase C (5) and alterations in intracellular Ca2+ (56). Similarly, cAMP may play a critical role in reestablishment of epithelial barrier function (8, 17) and in maintenance of endothelial barrier (34, 47). Others have reported that direct elevation of intracellular cAMP may decrease established barrier (6) or diminish TER after Ca2+ switch (4). The role of cAMP as a component of barrier recovery signal transduction is controversial. The results presented here seem to conflict with those of Balda et al. (4); however, these differences could arise from a number of different factors (e.g., agonist used and agonist concentration). For example, in our cell system, NECA may mobilize a distinct pool of cAMP efficiently coupled to PKA, as suggested by Barrett and colleagues (6). To expand on these observations, we studied the involvement of cAMP signal transduction in barrier recovery after Ca2+ switch, a valuable tool to assess mechanisms of tight junction assembly (21). Consistent with previous studies (54), agents that activate PKA (e.g., NECA; Fig. 1) do not significantly alter baseline permeability of confluent, high-resistance epithelia (such as T84 cells). Such findings are different, however, from those in high-permeability cell types, such as vascular endothelia. Indeed, multiple studies have demonstrated that activation of PKA results in a dynamic rearrangement of the endothelial cytoskeleton and a functional decrease in tight junction and/or adherens junction permeability (12, 18, 46). Conversely, PKA activation has been functionally implicated during tight junction reassembly, inasmuch as cellular and protein-bound cAMP levels were demonstrated to increase during the 1st h after Madin-Darby canine kidney cells were returned to normal Ca2+ levels after Ca2+ switch (9). Interestingly, we observed the greatest rate of resealing in the 1st h after return to normal Ca2+ levels (Fig. 3A). In our report, activation of PKA substantially promotes epithelial resealing (Fig. 1A), and, in parallel, inclusion of a specific PKA inhibitor resulted in a significant attenuation of resealing during Ca2+ switch (Fig. 1B). These studies further implicate PKA as an important component of tight junction assembly and as a mechanism of barrier recovery.

The cytoskeleton has been implicated in direct regulation of paracellular permeability. Perturbation of the actin cytoskeleton leads to increases in paracellular permeability, possibly via condensation of the perijunctional actin ring (38, 55) and phosphorylation of myosin light chain kinase (27). In addition, epithelial permeability and restitution are dependent on the cytoskeleton (41, 49). Several lines of evidence suggested a role for VASP in tight junction assembly. First, VASP is a major target for cyclic nucleotide kinases, including PKA (phosphorylation predominantly at Ser157) and PKG (10). Second, VASP has actin-binding and cross-linking capacities, both of which are conferred by the COOH-terminal region of the molecule (3) and, thus, could serve as a junctionally associated cytoskeletal link. Third, members of the Ena/VASP family of proteins have been implicated in processes that require actin remodeling. Localization of these proteins at actin-rich sites (such as focal adhesions and cell-cell junctions), coupled with their ability to promote actin nucleation, suggests that they are regulators of actin cytoskeleton dynamics (2, 7, 33), and, recently, VASP was demonstrated to be involved in actin reorganization and directional polymerization in epithelia (58). On the basis of these observations, we hypothesized a role for VASP in functional aspects of the tight junction reassembly. Initial studies indicated that VASP and the tight junction marker ZO-1 colocalize at the epithelial tight junction. Interestingly, the degree of barrier function may determine VASP association with tight junction complexes, since high-permeability epithelial monolayers (KB cells) revealed that VASP and ZO-1 colocalize only at regions of distinctly juxtaposed membranes. Further evidence was provided by coimmunoprecipitation, which indicated that ZO-1 and VASP are biochemically associated. It is not known whether ZO-1 and VASP directly associate. The molecular details of ZO-1 binding proteins are reasonably well understood and involve PDZ domains, a ~90-amino acid element present in three structurally related proteins PSD-95/SAP90, DLG, and ZO-1 (16). The PDZ-binding partners express a common (T/S)XV consensus sequence generally, but not without exception, on the COOH terminus (16). A close examination of the VASP amino acid sequence identifies at least three internal PDZ-binding domains, at amino acids 32-34 (SRV), 76-78 (TQV), and 322-324 (SSV). Alternatively, proteins containing SH3 domains, such as that in ZO-1, are known to bind with high specificity to proline-rich sequences (31). VASP contains a central proline-rich region, which has been demonstrated to mediate interactions with the SH3 domain of murine Abl and Src (1, 19), as well as with profilin (1). Important for the present work, this proline-rich domain in VASP (52% proline residues in amino acids 162-218) lies adjacent to the PKA binding site important in VASP conformational changes (i.e., Ser157), and transfection of truncations lacking this proline-rich region resulted in a significant inability of monolayers to reseal after Ca2+ switch. Thus it is possible that Ser157 phosphorylation could directly influence ZO-1-VASP interactions at this site, providing the basis for tight junction regulation by VASP. Further studies are necessary to ascertain whether VASP and ZO-1 directly interact or whether VASP functions as a bridge with other known ZO-1 binding partners.

A more clear understanding of VASP phosphorylation in the process of tight junction assembly was aided by utilization of reagents that selectively probe the structure-function aspects of phospho-VASP. First, a phosphospecific antibody directed against the PKA phosphorylation site of VASP (Ser157) allowed us to immunolocalize phospho-VASP after PKA activation and during Ca2+ switch. These studies determined that basal expression of phospho-VASP within the tight junction was indiscreet and morphologically diffuse. However, on PKA activation, phospho-VASP was rapidly localized to the tight junction. Moreover, the biochemical phosphorylation of VASP and the appearance of phospho-VASP at the tight junction after Ca2+ switch suggest that VASP, in its phosphorylated state, is an integral component of tight junction assembly. This occurrence is strengthened by the observation that phospho-VASP is absent from the tight junction in resting high-resistance monolayers (Fig. 6). Second, expression of truncated VASP protein lacking the EVH1 domain and the proline-rich region (including the preferred PKA binding site) resulted in diminished barrier recovery. Taken together, these observations suggest that the full-length VASP protein functions as a positive regulator of actin-based barrier function.

Although actin regulation by VASP is the focus of much recent work, the specific relationship between VASP binding and actin dynamics has not been fully elucidated, particularly with regard to VASP phosphorylation. In fact, this area is quite controversial in many regards, with reports of positive and negative regulatory aspects (35, 36). For example, VASP enhances Listeria motility by assembling actin filaments during migration within the cytoplasm of infected cells (11, 33). In addition, zyxin requires Ena/VASP proteins in cell-spreading experiments and is able to form actin-filled structures in an Ena/VASP-dependent manner (7, 15). Conversely, an increasing number of reports suggest that VASP may not always act to promote actin assembly and that the phosphorylation state of VASP is important in determining this function (25). For instance, phosphorylation of VASP in platelets closely parallels inhibition of actin-driven platelet aggregation (53). Other recent work has demonstrated that phosphorylation of VASP decreases its actin-nucleating activity and the ability to enhance polymerization (25).

Given the most recent evidence that phospho-VASP is likely a negative regulator of actin dynamics (25), several lines of evidence implicate phospho-VASP in structural relaxation of the actin cytoskeleton during junctional reassembly, resulting in accelerated recovery. First, the activation state of PKA directly parallels VASP phosphorylation and functional restitution of the epithelial tight junction. Second, junctional reassembly results in a time-dependent accumulation of phospho-VASP at epithelial tight junctions. Third, expression of VASP fragments lacking the PKA consensus domain decreases tight junction reassembly after Ca2+ switch, suggesting that structural changes associated with VASP phosphorylation relax cytoskeletal tension.

Taken together, such data suggest that VASP, and particularly phospho-VASP, may be more important in junctional reassembly during acute disruption, as occurs during PMN transmigration (37). Indeed, PMN transmigration has been demonstrated to depend on reorganization of the epithelial cytoskeleton (28), and PMN interactions with epithelia elevate intracellular cAMP through adenosine cross-talk pathways (40). Large epithelial disruptions resulting from PMN migration close through extension of lamellapodia enriched in the cytoskeletal elements F-actin, vinculin, and paxillin (51). It is possible, therefore, that phospho-VASP-mediated reassembly of the epithelial tight junction could provide an endogenous protective pathway for rapid resealing of epithelial barrier during acute disruption.


    FOOTNOTES

Address for reprint requests and other correspondence: S. P. Colgan, Brigham and Women's Hospital, Center for Experimental Therapeutics and Reperfusion Injury, 20 Shattuck St., Boston, MA 02115 (E-mail: colgan{at}zeus.bwh.harvard.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. Section 1734 solely to indicate this fact.

First published January 9, 2002;10.1152/ajpcell.00288.2001

Received 25 June 2001; accepted in final form 7 January 2002.


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DISCUSSION
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