Article |
Address correspondence to Estelle Sontag, Dept. of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9073. Tel.: (214) 648-2327. Fax: (214) 648-2077. E-mail: Estelle.Sontag{at}UTSouthwestern.edu
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Abstract |
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Key Words: PP2A; aPKC; ZO-1; occludin; claudin
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Introduction |
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Protein phosphatase (PP)2A enzymes are major Ser/Thr protein phosphatases. The core enzyme is a dimer containing a catalytic subunit (C) and a regulatory subunit (A), which can associate to a regulatory subunit (B). Several families of B subunits have been identified and modulate PP2A catalytic activity and substrate specificity (Sontag, 2001). Distinct B subunits contribute to targeting PP2A to defined intracellular domains and recruiting PP2A to signaling complexes, thereby ensuring its functional specificity (Sim and Scott, 1999; Sontag, 2001). Notably, the holoenzyme containing the B subunit is a major PP2A isoform involved in cell growth and cytoskeletal regulation in numerous cell types (Sontag, 2001). Here, we chose to undertake a detailed analysis of AB
C behavior in epithelial cells, a cell type for which PP2A properties and functions are poorly documented. We show that AB
C is targeted to the TJ complex and identify a novel role for PP2A in TJ regulation.
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Results |
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Inhibition of PP2A promotes TJ protein phosphorylation and TJ assembly
To explore the potential role of PP2A in TJ assembly, the distribution of ZO-1, occludin, and claudin-1 was compared by immunofluorescent microscopy and immunoblotting during Ca2+ switch experiments performed in untreated or OA-treated control MDCK cells, and in MDCK-Wt C cells (Figs. 5 and 6). Cells were Ca2+-starved overnight to induce TJ downregulation, resulting in internalization/redistribution of TJ proteins from the cell periphery to the cytosol, and then transferred to NC medium to induce TJ biogenesis. The Ca2+ switch initiates a rapid sorting of TJ proteins from the cytosol to the membrane; however, complete TJ stabilization and resealing, as measured by TER restoration, is only achieved >20 h after the calcium switch (Stuart and Nigam, 1995; Farshori and Kachar, 1999). 1 h after the Ca2+ switch, a portion of total TJ proteins had already migrated to the cell periphery in control MDCK cells, but this redistribution was largely inhibited after expression of Wt C (Fig. 5 A). The peripheral membrane staining for TJ proteins appeared more continuous after incubation of MDCK cells with OA. The critical role played by PP2A during early TJ biogenesis was even more clearly demonstrated during Ca2+ switch experiments performed in the absence of serum (Fig. 5 B). OA promoted, whereas expression of Wt C severely prevented the accumulation of TJ proteins at cell-cell contact sites. The inhibitory effect of expressed Wt C could be partially reversed by OA, reinforcing the idea that PP2A negatively regulates the initial sorting of TJ proteins to the membrane. 4 h after the Ca2+ switch, a complete junctional distribution of ZO-1 was achieved over the entire circumference of MDCK cells (Fig. 6 A). OA accelerated the formation of the TJ network. Expression of Wt C noticeably delayed the accumulation of TJ proteins at junctional areas, as revealed by the predominant cytosolic concentration and fragmentary staining of ZO-1 at cellcell boundaries. 24 h after the Ca2+ switch and thereafter, all the cells displayed the typical chicken wire staining pattern of ZO-1. However, cytosolic pools of TJ proteins were still visible at that stage in MDCK-Wt C cells. Significantly, in contrast to TJ proteins, changes in PP2A activity did not affect the redistribution of E-cadherin to areas of cellcell contact during AJ formation.
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TJ assembly also correlates with the development of TER. When MDCK-Wt C cells were switched from LC to NC medium to induce TJ resealing, they developed TER with much slower kinetics than control cells (Fig. 6 C). This delay in TER development was exacerbated when the Ca2+ switch was performed under serum-free conditions (LC to DC), validating our hypothesis that PP2A activity negatively regulates TJ assembly.
Effects of PP2A deregulation on F-actin
The complete reestablishment of the actin cytoskeleton architecture plays a crucial role during Ca2+-mediated junctional biogenesis, and the contraction of perijunctional F-actin critically regulates the TJ permeability barrier (Denker and Nigam, 1998). Thus, we addressed the hypothesis that PP2A regulates TJs via F-actin remodeling. As shown in Fig. 7, we could not observe any significant difference in the organization of F-actin in MDCK cells subjected to a Ca2+ switch, whether they were treated with OA, or transiently or stably expressing HA-tagged Wt C. Notably, subconfluent MDCK cells expressing high levels of transfected Wt C subunits exhibited a flattened, irregular shape with occasional lamellipodia, that was somewhat reminiscent of the morphology of cells expressing dominant-negative mutants of atypical protein kinase C (aPKC) (Suzuki et al., 2001). Overall, OA treatment or Wt C expression did not dramatically affect the appearance of the cortical F-actin bundles (see "apical" sections) and stress fibers (see "basal" sections) present in confluent cells cultured in NC medium. However, now and then, the pattern of stress fibers appeared interwoven or less dense in confluent MDCK-Wt C cells.
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Discusssion |
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So far, the evidence for a role for Ser/Thr phosphatases in TJ regulation is fragmentary, and derives entirely from studies utilizing OA and calyculin A, which inhibit PP2A/PP1 enzymes in vivo in a dose- and time-dependent manner. Interestingly, these inhibitors are naturally occurring toxins that modulate intestinal paracellular permeability and are responsible for diarrheic shellfish poisoning in humans (Tripuraneni et al., 1997; Okada et al., 2000). As reported previously in other epithelial cells (Singer et al., 1994; Tripuraneni et al., 1997; Okada et al., 2000), we observed that high concentrations of OA or prolonged incubation of MDCK cells with this inhibitor induced cell rounding and TJ leakiness. Under these conditions, not only PP2A but also PP1 enzymes became inhibited, and a direct role for PP2A in TJ regulation cannot be meaningfully evaluated. Instead, lower OA concentrations more selective for PP2A did not appreciably alter the organization of the TJ network and the resistance and paracellular permeability of MDCK cells grown in NC medium, in agreement with previous studies (Pasdar et al., 1995; Tripuraneni et al., 1997; Okada et al., 2000). At first sight, this may suggest that PP2A does not regulate TJs once formed. However, enhanced PP2A activity induced dephosphorylation of membrane-associated TJ proteins, leading to decreased TER and increased TJ permeability. Based on previous studies pointing to an important role for occludin dephosphorylation in promoting TJ opening (Farshori and Kachar, 1999; Clarke et al., 2000; Simonovic et al., 2000), reduced levels of TJ-associated, phosphorylated occludin may contribute in part to enhanced TJ leakiness in MDCK-Wt C cells. The fact that OA can inhibit the effects of expressed Wt C suggests that PP2A-dependent changes in the phosphorylation state of TJ-bound proteins modulate the dynamic opening/closing of mature TJs. Many signaling molecules mediate changes in TJ barrier properties via remodeling of the actin cytoskeleton (Denker and Nigam, 1998). Yet, we were unable to demonstrate that changes in PP2A activity induce clear-cut effects on F-actin organization under our experimental conditions. Likewise, OA does not significantly affect F-actin distribution in carcinoma cells (Strnad et al., 2001). However, the effects of OA on F-actin are dose- and time-dependent (Leira et al., 2001); high OA concentrations consistent with PP1 inhibition disrupt F-actin (Fiorentini et al., 1996).
OA promoted the phosphorylation and recruitment of TJ proteins to TJs during junctional assembly. Reciprocally, expressed Wt C induced the accumulation of soluble, dephosphorylated forms of TJ proteins, which correlated with a severe delay in the sorting of TJ proteins from the cytosol to TJs, and retardation in TER development. The observation that OA induces the membrane translocation of TJ proteins in LC medium and reverses the effects of Wt C in the absence of serum is consistent with the hypothesis that inhibition of PP2A promotes TJ assembly. Thus, the phosphorylation state of TJ proteins is controlled by PP2A and is closely linked to their ability to redistribute to the membrane during junctional biogenesis. It has been proposed that the nucleation of AJ assembly by cadherin/catenin complexes controls TJ assembly (Gumbiner et al., 1988). Although PP2A C subunit associates with and stabilizes the ß-catenin/E-cadherin complex in immature blastocysts (Gotz et al., 2000), we were unable to colocalize or coimmunoprecipitate AB
C and E-cadherin in polarized MDCK cells. The accumulation of E-cadherin at regions of cellcell contact also proceeded normally despite PP2A deregulation, suggesting that PP2A-dependent defects in TJ assembly do not occur secondary to abnormalities in AJ formation. Together with the observations that AB56C, but not AB
C, regulates ß-catenin in the Wnt signaling pathway (Li et al., 2001), and that AB
C, but not AB56C, associates with TJ proteins, our findings suggest that the regulation of TJs by PP2A likely involves an E-cadherinindependent pathway (Stuart and Nigam, 1995) controlled by AB
C. However, the hierarchical regulation of junctional complexes is not absolute but rather multifaceted, as inhibition of cadherin adhesion can exert both negative and positive effects on TJ biogenesis in MDCK cells (Troxell et al., 2000). It is thus possible that distinct PP2A holoenzymes differentially affect separate signaling pathways that converge on TJs.
The results from our inhibitor studies indicate that aPKC participates in the regulation of ZO-1, occludin, and claudin-1 by PP2A. OA induced a 2.7-fold activation of membrane-bound aPKC, in agreement with the finding that membrane-associated PKC activity more than doubles during TJ assembly (Stuart and Nigam, 1995). Our conclusions support those of previous reports showing the importance of aPKC-mediated TJ protein phosphorylation for Ca2+-induced TJ assembly (Stuart and Nigam, 1995; Suzuki et al., 2001). PP2A associated with both soluble and membrane-bound aPKC and modulated aPKC activity and distribution. Thus, PP2A may be part of and directly regulate the aPKC/Par-3 signaling complex that is involved in regulation of TJ formation and cell polarity. The aPKC/Par-3 complex is tethered to TJs via JAM. It is noteworthy that, as with Wt C, overexpression of a JAM mutant disrupts the localization of aPKC and ZO-1 without significantly affecting the localization of E-cadherin (Ebnet et al., 2001).
In conclusion, we demonstrate that ABC is recruited to and is a novel component and regulator of the TJ signaling complex. The regulation of TJs by PP2A is dependent on functional aPKC. We establish PP2A as a novel regulator of aPKC and identify the TJ proteins, ZO-1, occludin, and claudin-1, as targets of PP2A/aPKC signaling in epithelial cells.
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Materials and methods |
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Cell culture and characterization of stable MDCK cell lines
All experiments were performed in the highly polarized MDCK strain II D5 clonal cell line (Brewer and Roth, 1991). Cells were maintained on plastic dishes in DME (Gibco BRL) containing 10% FBS (Hyclone). For most experiments, cells were plated at high density on Transwell filters (Costar) and cultured until complete polarization, as verified by measuring TER. Cells were transfected using Lipofectamine Plus reagent (Gibco BRL). Stable clones were obtained after selection with 800 µg/ml geneticin (Gibco BRL), analyzed for expression of the transfectant, and pooled together to minimize the effects of clonal variations. Seven separate pooled populations of stable transfectants were utilized throughout our studies. The expression level of transfected proteins was constantly monitored by immunofluorescence and immunoblotting. Immunoblotting indicated that HA-tagged Wt C subunit was expressed at 3050% of the levels of endogenous C in stable MDCK cell lines, as previously reported in NIH 3T3 cells (Ogris et al., 1997). HA-tagged B
subunits were expressed at
20% of the levels of endogenous B
. Stable MDCK cells transfected with the pcDNA3.1 vector were used as controls (control MDCK cells) and behaved like nontransfected cells in our experiments. Sodium butyrate (2.55 mM; Sigma-Aldrich) was added in the culture medium for
16 h before each experiment to enhance expression of the transfected proteins, and had no detrimental effects on any of the parameters measured in our studies, as reported previously (Brewer and Roth, 1991).
Calcium switch experiments
To induce rapid TJ disassembly, monolayers of MDCK cells grown in normal Ca2+ (NC) medium (DMEM + 10% FBS; 1.8 mM Ca2+) were incubated in LC medium (Ca2+-free S-MEM; Gibco BRL) containing 1 mM EGTA. For prolonged Ca2+ removal, cells were incubated overnight in LC medium containing 1% dialyzed FBS. For the Ca2+ switch, Ca2+-starved cells were transferred to either NC or DC (DMEM + 1% dialyzed FBS) medium. If indicated, OA (Alexis Biochemicals) or the vehicle (100% DMSO) was added to the medium during the Ca2+ switch.
Transepithelial resistance measurements
Cells were plated at confluency and grown on sixwell Transwell filters in NC medium. TER values were measured in duplicate wells using an EndohmTM voltohmmeter (World Precision Instruments). TER values (.cm2) were normalized to the area of the monolayer (filter), and calculated by subtracting the blank values (
18
.cm2) from the filter and the bathing medium. All cell culture media were supplemented with 25 mM Hepes, pH 7.4, and the integrity and cell density of the monolayers were carefully monitored during TER measurement studies.
Paracellular diffusion measurement
Cells were grown on 6.5-mm Transwell filters until complete polarization. TJ leakiness was assessed by measuring the diffusion of [3H]-inulin (Amersham Biosciences) and [3H]-mannitol (NENTM Life Science Products, Inc.) across the membrane in monolayers of equivalent cell density (Wong and Gumbiner, 1997). The tracer diffusion was examined by replacing the apical compartment medium by fresh NC medium containing the tracer (2.5 µCi/ml), and the basal compartment medium with the same medium without tracer. When indicated, OA or the vehicle (DMSO) was added in both basal and apical bathing media. The compartment media were collected 60 min afterwards, and counted by liquid scintillation.
Confocal microscopy
Cells grown on Transwell filters or on glass coverslips were fixed with methanol for 5 min at -20°C, and labeled sequentially for 1 h with primary and secondary antibodies at a 1:100 dilution (Sontag et al., 1995). For the visualization of F-actin, cells were fixed for 20 min with 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and labeled for 30 min with FITC-labeled phalloidin (Sigma-Aldrich). The samples were mounted using Fluoromount-G (Fisher) and examined on a Leica TCS SP confocal microscope using a 63x objective. Images (16 x-y or x-z sections across cells) were directly captured, saved and transferred to Adobe Photoshop® 5.5 for printing. The specificity of the labeling was verified by omitting first or second antibodies during the staining procedures, and by using pre-immune sera and antibodies that had been preadsorbed with the corresponding antigen.
Electron microscopy
Cryosections (80 nm) from normal human adult colon fixed in 1% glutaraldehyde/4% paraformaldehyde were picked up using a 1.8-mm loop with a droplet of frozen 2.3 M sucrose, thawed, moved onto Formvar-covered and carbon-coated glow-discharged 100 mesh copper grids and stored on buffer. Immunogold labeling of the cryosections was performed by the Tokuyasu method (Tokuyasu, 1986) by which the grids are moved through series of droplets containing blocking agents, antibodies and are ultimately coated with a layer of 0.2% uranyl acetate in 2% methylcellulose. The cryosections were incubated for 30 min each with serial dilutions of the primary antibody in PBS containing 5% milk, and then with protein A gold (10 nm diameter; 1:70 dilution; a gift from G. Posthuma, Utrecht University Medical Center, The Netherlands). The cryosections were examined using a JEOL 1200EX Transmission Electron Microscope operating at 120kV. Electron micrographs were taken using Eastman Kodak SO-163 electron image film.
Cell fractionation and Western blotting
To prepare detergent-soluble and -insoluble fractions, confluent cells were washed in PBS, harvested in 400 µl buffer 1 (25 mM Tris, 150 mM NaCl, 1% NP-40, 4 mM EDTA, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 1 µM OA, pH 7.4) containing a cocktail of protease inhibitors (Roche) and incubated for 30 min at 4°C. The extracts were then centrifuged for 30 min at 4°C in a microfuge to yield a soluble fraction (supernatant), after which the remaining insoluble fraction (pellet) was harvested in the same buffer, sonicated to disrupt protein aggregates, then recentrifuged to eliminate insoluble material. To prepare membrane fractions, cells were washed in PBS and incubated for 5 min at room temperature in buffer S (0.25 M sucrose, 1 mM imidazole, 5 mM MgCl2). The medium was aspirated and cells were harvested in buffer S containing 1 µM OA, 1 mM DTT and a cocktail of protease inhibitors, incubated on ice for 15 min then dounce homogenized. Cells were centrifuged at 800 g at 4°C to pellet nuclei, after which the supernatant was centrifuged at 4°C for 45 min at 100,000 g in a Beckman TL-100.3 rotor. The supernatant (cytosol) was collected and the remaining pellet (membrane fraction) was resuspended in buffer 1 and sonicated. Equivalent aliquots of proteins from the cell fractions determined using Bio-Rad protein assay kit were resolved by SDS-PAGE on 5% (for ZO-1), 8% (for occludin, E-cadherin, and aPKC) and 12% (for PP2A subunits and claudin-1) polyacrylamide gels, and analyzed by immunoblotting for the presence of PP2A subunits or junctional proteins. Immunoreactive proteins were detected using SuperSignal Chemiluminescence substrates (Pierce Chemical Co.).
Immunoprecipitation
Cytosolic/membrane fractions were normalized for protein concentration and volume, after which the buffer was adjusted to 150 mM NaCl and 1% NP-40. After preclearing, total, detergent-soluble/insoluble, or cytosolic/membrane fractions were incubated overnight at 4°C with either the indicated antibodies (7 µl antibody/ml cell extract), or no antibody to assess nonspecific binding. The immunoprecipitates were collected using either protein A Sepharose or G PLUS-agarose beads (Santa Cruz Biotechnology), washed extensively in buffer 1, and resuspended in Laemmli sample buffer. In some experiments, the immunoprecipitations were carried out with HA-tagged antibody-coupled affinity matrix (Covance) and control nontransfected cells were used in parallel assays to verify the specificity of the immunoprecipitations (Goedert et al., 2000). Equivalent aliquots of the immunoprecipitates were analyzed by SDS-PAGE on 415% gradient Ready gels (Bio-Rad Laboratories) and transferred to nitrocellulose. The blots were cut into strips and simultaneously immunoblotted with antibodies directed against PP2A subunits, PP1
, and junctional proteins to allow for comparative analysis of the relative amounts of immunoprecipitated material in each condition. In other experiments, the colon and kidney from a 10-wk old rat was minced, homogenized in buffer 1 with a tissue grinder and centrifuged at 13,000 g for 15 min to remove insoluble material. Aliquots of the supernatants were immunoprecipitated with mouse anti-occludin antibody and analysed as described above.
Analysis of TJ protein phosphorylation
MDCK cell detergent-insoluble fractions were immunoprecipitated as described above with antiZO-1, -occludin, or -claudin-1 antibodies. The immunoprecipitates were washed and resuspended in P buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT, 1 mM sodium fluoride, 100 µM sodium orthovanadate). Phosphorylation of TJ proteins was performed for 1 h at 30°C by adding 10 µg phosphatidylserine, a gift from P. Sternweis (UT Southwestern, Dallas TX), 1 µg recombinant human aPKC
(Calbiochem), and 100 µM [
-32P]ATP (5 µCi) per reaction. The tubes containing phosphorylated TJ proteins were transferred on ice and carefully divided into 15-µl aliquots. Either 100 nM purified AB
C (Goedert et al., 2000), 100 nM AB
C preincubated on ice for 20 min with 1 µM OA, or buffer alone, were added into the reaction mixtures. The samples (25 µl) were incubated for another 30 min at 30°C, after which the reactions were terminated by addition of 3x sample buffer for SDS-PAGE. The samples were boiled for 5 min then simultaneously analyzed by SDS-PAGE on 4-20% gradient gels (Bio-Rad Laboratories), followed by autoradiography. In other experiments, ZO-1, occludin, and claudin-1 were immunoprecipitated from membrane fractions, resolved by SDS-PAGE and analysed by immunoblotting using rabbit anti-phosphoserine antibody. The blots were reprobed with anti-ZO-1, occludin, and claudin-1 antibodies. Phosphorylation of PKC
was examined by immunoblotting using p-nPKC
(Thr410) antibody (Standaert et al., 1999a). In parallel, aliquots of cell fractions were resuspended in AP buffer (50 mM Tris, pH. 7.4, 50 mM NaCl, 1 mM MgCl2, 1 mM DTT, 0.1% NP-40, and cocktail of protease inhibitors) and incubated for 1 h at 30°C with or without alkaline phosphatase (20 U/sample; Roche) before being analyzed by immunoblotting.
aPKC activity assays
aPKC activity was measured in immunoprecipitates as described previously (Standaert et al., 1999a). After washing in P buffer, aPKC immunoprecipitates were incubated for 15 min at 30°C with 50 µl of P buffer containing 50 µM ATP, 1 µCi [-32P]ATP, and 5 µg serine analogue of PKC
-pseudosubstrate (BioSource International), a selective aPKC substrate. The reactions were performed in the presence or absence of 10 µM PKC
/
pseudosubstrate (BioSource International), as described previously (Sontag et al., 1997). The reaction mixtures were spotted onto P-81 phosphocellulose paper and washed with 75 mM phosphoric acid. Incorporation of 32P was determined by liquid scintillation counting.
PP2A activity assays
Aliquots of cell extracts were assayed for PP2A activity for 6 min at 30°C using the RRREEE(pT)EEE phosphopeptide (Biosynthesis, Inc.) as a substrate. Dephosphorylation of the peptide was determined by measuring the release of Pi using a quantitative colorimetric assay (Sontag et al., 1999).
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Footnotes |
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* Abbreviations used in this paper: AJ, adherens junction; aPKC, atypical PKC; JAM, junctional-adhesion molecule; LC, low Ca2+; MDCK, Madin-Darby canine kidney; NC, normal Ca2+; OA, okadaic acid; PP, protein phosphatase; TER, transepithelial resistance; TJ, tight junction; ZI, PKC
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Acknowledgments |
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E. Ogris serves as a consultant to Upstate Biotechnology, Inc. This work was supported by National Institutes of Health grant AG18883 to E. Sontag, and a grant from the Austrian Science Foundation (FWF, P13707-MOB) to E. Ogris.
Submitted: 26 June 2002
Revised: 22 July 2002
Accepted: 22 July 2002
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