Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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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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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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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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 · cm2) and
low-resistance (KB cells, TER < 50
· 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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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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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 · 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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DISCUSSION |
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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.
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FOOTNOTES |
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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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