A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion

Jiuming Ye1, Tatsuo Tsukamoto1, Adam Sun2, and Sanjay K. Nigam1

1 Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and 2 Renal Division, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The integrity of the tight junction (TJ), which is responsible for the permeability barrier of the polarized epithelium, is disrupted during ischemic injury and must be reestablished for recovery. Recently, with the use of an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney cells, TJ proteins such as zonula occludens-1 (ZO-1) were shown to reversibly form large complexes and associate with cytoskeletal proteins (T. Tsukamoto and S. K. Nigam, J. Biol. Chem. 272: 16133-16139, 1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability barrier following ATP repletion as measured by transepithelial electrical resistance and mannitol flux, marked alterations in the subcellular localization of occludin by immunofluorescent analysis, and decreased solubility of ZO-1 and other TJ proteins by Triton X-100 extraction assay, suggesting that lowering intracellular calcium potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that decreased solubility may partly result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) appeared to cause a dissociation of ZO-1-containing complexes from the cytoskeleton, sucrose gradient analyses of the solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium plays an important facilitatory role in the reassembly of the TJ damaged by ischemic insults. Calcium appears to be necessary for the dissociation of TJ-cytoskeletal complexes, thus permitting functional TJ reassembly and paracellular permeability barrier recovery.

cytoskeleton; ischemia; injury; recovery; signaling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TO CARRY OUT VECTORIAL transport of solutes and water, polarized epithelial cells such as renal tubular cells must establish and maintain a permeability barrier between two different cellular compartments (34). The maintenance of this permeability barrier is largely achieved by specialized intercellular junctions, particularly the tight junction (TJ). The TJ is located at the most apical part of the epithelial lateral membrane, providing a "fence" separating apical and basolateral domains and a "gate" modulating paracellular transport (6, 30). In recent years, a number of proteins have been localized at the TJ. Among the better characterized of these are the highly homologous proteins zonula occludens (ZO)-1 (20, 39), ZO-2 (6, 8, 16, 21), and the newly characterized ZO-3, also known as p130 (17). These cytosolic proteins are part of the submembrane plaque of the TJ and may be directly or indirectly associated with the actin-based cytoskeleton (13, 14, 19, 22, 26, 45). Other identified peripheral TJ proteins include 7H6 antigen, cingulin, symplekin, and AF-6, although these are less well characterized (5, 23, 49, 50). Other candidates include phosphoproteins of ~330 and 65 kDa (41). Occludin is the best-characterized integral membrane protein of the TJ and is believed to play an essential role in the fence and gate functions (12, 30). However, studies using occludin gene disruption analysis and the recent identification of claudin-1 and -2 indicate that multiple integral membrane proteins likely constitute the TJ (11, 35). Thus it is likely that the macromolecular structure of the TJ and the interactions among these TJ proteins are highly complex.

Although the structural and functional roles of several TJ proteins have been investigated in some detail, there is only limited information on how the TJ complex is assembled and regulated (6). These questions are not only significant in the field of epithelial morphogenesis and development, in which the permeability barrier must be formed de novo (8), but are also clinically relevant in the context of recovery from acute epithelial injury. For example, in acute kidney failure due to ischemia, the permeability barrier of tubular epithelial cells is disrupted and must be reestablished through reassembly of the TJ for the kidney to function again (10, 25). However, little is known about TJ assembly in these contexts.

Epithelial injury caused by ischemia and the subsequent reperfusion/reoxygenation leads to mispolarization of membrane proteins, perturbation of the actin cytoskeleton, disruption of the permeability barrier, and diminished capacity to fold and assemble secretory proteins in the endoplasmic reticulum (24, 31, 45). These changes have been reproduced in cell culture models for hypoxia-reoxygenation injury using agents that deplete cellular ATP, which allows for analysis of the molecular events underlying ischemic injury (27, 28). Recently, we studied the biochemical basis of the disruption of the TJ with a ATP depletion-repletion model in Madin-Darby canine kidney (MDCK) cells. Treatment of the cells with a combination of inhibitors of glycolysis (2-deoxy-D-glucose) and oxidative phosphorylation (antimycin A) demonstrated distinct and reversible changes in the function, morphology, and biochemistry of the TJ (45). For example, TJ disassembly was accompanied by the association of TJ proteins into large macromolecular complexes, movement of TJ proteins into an insoluble pool, and an increased association between TJ proteins and the actin-based cytoskeleton. We have recently demonstrated that a tyrosine kinase plays a key role in TJ reassembly in this model (45a).

Nevertheless, the regulatory mechanisms underlying TJ disassembly-reassembly after ATP depletion-repletion (as well as in other injurious settings) remain largely unknown. For many years, the role of intracellular calcium in epithelial injury and repair has been examined and debated (47). Because intracellular calcium has been shown to be necessary for TJ assembly in the "calcium switch" model for polarized epithelial biogenesis (33, 42, 43), we have now explored the role of intracellular calcium in the reestablishment of the permeability barrier and several biochemically defined steps in TJ reassembly following ATP depletion-repletion in MDCK cells. In this study, we demonstrate that the lowering of intracellular calcium inhibits the functional as well as the morphological recovery of TJ, partly because of altered interactions between the TJ proteins and the cytoskeleton. Our data indicate that, in this model of ischemic injury to epithelial cells, intracellular calcium is required at one or more steps for the dissociation of large, insoluble complexes involving TJ and cytoskeletal proteins that accumulate following ATP depletion.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials and cell culture. MDCK cells (strain I), provided by Dr. K. Matlin of Massachusetts General Hospital (Boston, MA), were maintained in DMEM (GIBCO BRL, Grand Island, NY) that was supplemented with 5% FCS as previously described (45). Anti-ZO-1 hybridoma R40.76 was kindly provided by Dr. D. Goodenough (Harvard). Anti-occludin polyclonal antibody was purchased from Zymed (South San Francisco, CA). Anti-fodrin antibody was a generous gift from Dr. J. Hartwig (Harvard). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM and 5,5'-dimethyl-BAPTA-AM were purchased from Molecular Probes (Eugene, OR). All other reagents used in these experiments were of analytical grades.

ATP depletion-repletion and calcium chelation. Depletion of ATP was achieved rapidly in MDCK cells by using a combination of glycolytic (2-deoxy-D-glucose) and oxidative (antimycin A) inhibitors, as previously described (45). Briefly, confluent monolayers were washed three times with PBS and then exposed to Dulbecco's PBS containing 1.5 mM CaCl2, 2 mM MgCl2, 2 mM 2-deoxy-D-glucose, and 10 µM antimycin A (Sigma, St. Louis, MO) for 1 h. The ATP-depleted cells were then replenished with normal growth medium (ATP repletion). Samples designated as control were obtained from cultures grown in standard growth medium. For intracellular calcium chelation, BAPTA-AM at the indicated concentrations was added to the ATP depletion buffer 30 min before ATP repletion.

Transepithelial electrical resistance and paracellular permeability for mannitol. MDCK cells were plated at confluent density (~2 × 105 cells/cm2) on polycarbonate filters (Transwells; Corning Costar, Cambridge, MA) and allowed to establish tight monolayers over 48 h before ATP depletion-repletion with metabolic inhibitors. Transepithelial electrical resistance (TER) was measured with a Millipore electrical resistance system (Millipore, Bedford, MA) as previously described (42, 45). Measurements are expressed as percent of initial value after subtraction of background readings. Paracellular permeability for mannitol was determined as described (1). Briefly, [3H]mannitol (1 µCi/ml, 30 Ci/mmol) (DuPont NEN, Wilmington, DE) was added to the apical side of the monolayers under the indicated conditions (control, ATP depletion, ATP depletion-repletion, and BAPTA-AM treated). After a brief incubation period at room temperature (20 min), the unilateral flux of mannitol was determined by serially measuring the amount of radioactive tracer present in the basal compartment of Transwell plates.

Measurement of intracellular calcium. Intracellular calcium was measured fluorometrically (SLM AB2) in fura 2-loaded cells as previously described (42, 43). To reduce autofluorescence, DMEM was freshly prepared based on the composition of DMEM provided by the manufacturer (GIBCO BRL) without phenol red, vitamins, and amino acids. Monolayers of MDCK cells grown on glass coverslips were first incubated with 5 µM fura 2 for 60 min at room temperature. The coverslips were then attached to the chamber and incubated with the indicated solutions. The fluorescence was measured at 510 nm after alternating exposures to excitation at 340 and 380 nm. All calcium measurements were performed at 37°C. Calibration was carried out at the end of each experiment with the calcium ionophore, ionomycin (10 µM) in the presence of DMEM or ATP depletion solution either containing 1.5 mM calcium (maximum ratio) or 2 mM EGTA without calcium (minimum ratio), and intracellular calcium levels were calculated as described (15, 43).

Immunocytochemistry. Confluent monolayers of MDCK cells grown on glass coverslips were subjected to ATP depletion-repletion in the presence or absence of BAPTA-AM, followed by immunostaining as previously described (41, 45). Briefly, after cells were washed three times with PBS, cells were fixed in 1% paraformaldehyde for 15 min, permeabilized in PBS-0.075% saponin, and then incubated for 1 h with primary antibodies at room temperature. After they were washed, the coverslips were incubated in tetramethylrhodamine isothiocyanate- or FITC-conjugated secondary antibody (Jackson Laboratories, West Grove, PA) and mounted in Gelvatol. Coverslips were observed with a laser scanning confocal system (Bio-Rad, MRC 1024) coupled to a Zeiss Axioskop microscope through a ×100 oil-immersion objective. Images were processed using Laser Sharp (Bio-Rad) and PhotoShop softwares (Adobe, CA).

Western immunoblot analysis. Electrophoresed proteins were transferred to nitrocellulose filters (MSI, Westboro, MA) by electroblotting. After membranes were blocked with a buffer containing 2% fat-free dry milk, 1% Triton X-100, 50 mM Tris · HCl (pH 7.4), and 10 mM EDTA, membranes were incubated with primary antibodies for 1 h at room temperature. After filters were washed, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratories) and then developed using the SuperSignal chemiluminescent, HRP-conjugated substrate system (Pierce, Rockford, IL).

Detergent-salt extraction assay. Confluent monolayers of cells were subjected to detergent-salt extraction as previously described (42, 45). After ATP depletion-repletion, monolayers grown on six-well plates were rinsed three times with ice-cold PBS and then extracted by overlaying the cells with a buffer containing 0.5% Triton X-100, 100 mM NaCl, 10 mM Tris · HCl (pH 7.4), and 300 mM sucrose plus a protease inhibitor cocktail (20 mg/ml each of phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A, and antipain) for 20 min at 4°C on a gently rocking platform. The extract fraction was completely aspirated and collected, and the residue fraction was separated and dissolved in the same volume of 2× sample buffer. Aliquots of both fractions, two volumes of the extract fraction and one volume of residue fraction at each condition, were analyzed by SDS-PAGE and Western immunoblotting.

Sucrose density gradient analysis. MDCK cells were lysed in a buffer containing 1% Triton X-100, 0.5% deoxycholate, 0.2% SDS, 100 mM NaCl, 10 mM HEPES (pH 7.5), 2 mM EDTA, and 1 mM sodium orthovanadate, plus protease inhibitor cocktail, and layered on top of linear 5-20% sucrose gradients prepared in the lysis buffer without detergents. The gradients were centrifuged at 32,000 rpm for 24 h in a Beckman SW40TI ultracentrifuge rotor at 4°C. Seventeen fractions were collected using an Auto Densi-Flow IIC gradient fractionator (Buchler Instruments, Lenexa, KS) and analyzed by SDS-PAGE and Western immunoblotting (45). The gradients were standardized with molecular mass markers ranging from 80 to 669 kDa (Sigma).

Immunoprecipitations. Confluent MDCK cell monolayers were subjected to ATP depletion-repletion and BAPTA-AM treatment as described in ATP depletion-repletion and calcium chelation and then scraped into a buffer containing 1% Triton X-100, 1% NP-40, 150 mM NaCl, 20 mM HEPES (pH 7.5), 300 mM sucrose, 1.5 mM MgCl2, 1 mM EGTA, and protease inhibitor cocktail as described in Detergent-salt extraction assay (above) and incubated in the buffer for 30 min at 4°C with gentle mixing. The insoluble materials were separated by centrifugation at 14,000 g for 30 min at 4°C. After they were precleared, the supernatants were incubated for 12 h with ZO-1 hybridoma supernatant or rat nonimmune serum at 4°C, followed by incubation with protein G Sepharose beads (Zymed) for 60 min at 4°C. Beads were then collected by brief centrifugation, washed five times with the immunoprecipitation buffer, boiled in 2× sample buffer, and subjected to SDS-PAGE, immunoblotting with antibodies to ZO-1, ZO-2, and fodrin.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular calcium chelation markedly inhibits the recovery of TER and paracellular flux after ATP depletion-repletion. Intracellular calcium plays a critical role in TJ biogenesis in the calcium switch model for junction biogenesis (6, 33, 42, 43). We hypothesized that intracellular calcium might also play a role in TJ reassembly following ATP depletion-repletion of MDCK cell monolayers. Treatment of confluent monolayers with a combination of antimycin A and 2-deoxy-D-glucose (ATP depletion) induced a rapid decrease of TER in monolayers grown on Transwell filters. This drop in TER was readily reversible by switching to normal growth medium (ATP repletion), as evidenced by the return to near normal (~80%) levels of TER following 3 h of ATP repletion (Fig. 1). This is consistent with previous findings (45) and indicates that the model system is reversible and that cell survival was not affected over the period of the experiment.


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Fig. 1.   Intracellular calcium chelator, 1,2-bis(2-aminophenoxy)- ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, markedly inhibits the recovery of transepithelial electrical resistance (TER) after ATP depletion-repletion. Madin-Darby canine kidney (MDCK) cell monolayers were ATP depleted for 1 h with 2 mM 2-D-deoxyglucose and 10 µM antimycin A. BAPTA-AM (25 or 50 µM) was added during the last 30 min of ATP depletion. ATP repletion was achieved by replenishing cells with normal growth medium after ATP depletion and BAPTA-AM treatment. Values of TER at the beginning of the experiments were taken as 100%. TER measurements are means ± SD of 3 experiments, with the data calculated as a percentage of initial values corrected for background. , Control; , 25 µM BAPTA-AM; open circle , 50 µM BAPTA-AM.

To investigate the role of intracellular calcium in the reassembly of the TJ following ATP depletion-repletion, we employed the cell-permeant calcium chelator BAPTA-AM. When monolayers were treated with BAPTA-AM for 30 min (during the second half of ATP depletion), we consistently observed a significant delay in the recovery of TER following ATP repletion (Fig. 1). Higher concentrations of BAPTA-AM (50 µM) exhibited a more significant inhibitory effect than a lower concentration (25 µM), indicating a dose dependency of this effect. In addition, 5,5'-dimethyl-BAPTA-AM, a more potent analog of BAPTA-AM, was capable of inhibiting the recovery of TER even after 7 h of ATP repletion (data not shown). Consistent with our prior work, treatment of BAPTA-AM in the absence of ATP depletion had a minimal effect on TER of established, confluent MDCK cell monolayers (43). We observed that full recovery of TER following ATP repletion in the BAPTA-AM-treated cells usually occurred within 24 h (data not shown). In addition, we consistently observed that TER "overshoots" the baseline after 5-7 h of ATP repletion in MDCK cells (Fig. 1). These values return to baseline within 12 h of ATP repletion. A similar observation has been described when the calcium switch model was used to study the assembly or biogenesis of TJs (7, 29). This may reflect some transient remodeling of the TJ late in the reassembly process.

Although the function of the epithelial permeability barrier is most frequently measured by TER, recent reports raise the possibility that functional dissociation of paracellular permeability and TER can occur in certain instances (2). Therefore, we measured paracellular flux during ATP depletion-repletion and in the presence of BAPTA-AM. As shown in Fig. 2, paracellular flux significantly increased following ATP depletion and subsequently returned to near initial levels after 3 h of ATP repletion. Treatment with BAPTA-AM significantly inhibited the return of paracellular permeability back to the initial level. These results are consistent with the TER data described above indicating that BAPTA-AM has an effect on the overall functional recovery of the epithelial cell monolayer following ATP depletion-repletion.


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Fig. 2.   BAPTA-AM significantly inhibits the reestablishment of the paracellular permeability barrier following ATP depletion-repletion. After ATP depletion-repletion and treatment with BAPTA-AM (50 µM), [3H]mannitol (1 µCi/ml in Dulbecco's PBS containing 1.5 mM CaCl2 and 2 mM MgCl2) was added to the apical side of the MDCK cell monolayers, which were grown on polycarbonate filters (Transwells). The amount of tracer that migrates across the monolayer and into the lower chamber was determined by scintillation counting of the basal compartment. Measurements after 1 h of ATP depletion were taken as 100%, and values are means ± SD of 3 experiments. * Statistically significant difference (P < 0.005).

Intracellular calcium levels in MDCK cells remain stable during ATP depletion-repletion but is lowered by BAPTA-AM. Next, we monitored the intracellular calcium level following ATP depletion and repletion in the presence or absence of BAPTA-AM in MDCK cell monolayers. Because of our previous observation in the calcium switch model that there is an early rise of intracellular calcium that quickly returns to baseline within minutes of calcium switch (42, 43), we chose to monitor intracellular calcium continuously. We did not observe any significant changes in the overall intracellular calcium level within the first 1 h following ATP depletion alone or the first 1 h following ATP repletion as shown in Fig. 3. This lack of calcium response induced by ATP depletion alone in MDCK cells has been previously observed by others (37, 46). On the other hand, intracellular calcium decreased rapidly during ATP depletion in the presence of BAPTA-AM (50 µM) and remained low following ATP repletion for 1 h (Fig. 3). Thus BAPTA-AM appears to be chelating intracellular free calcium, which has a direct effect on functional recovery of the TJ. Hence, at the very least, these data support a permissive role for intracellular calcium in TJ recovery, although this does not exclude a regulatory role. To gain insight into the mechanisms underlying the involvement of calcium in TJ reassembly within this context, we sought to perform biochemical and immunocytochemical analyses.



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Fig. 3.   Intracellular calcium concentration ([Ca2+]i) level remains stable during ATP depletion-repletion in MDCK cells but is lowered by BAPTA-AM. Intracellular calcium was measured with fura 2-loaded MDCK cells following ATP depletion and repletion in the presence or absence of BAPTA-AM (50 µM). A, top: intracellular calcium level after ATP depletion and repletion. At indicated time points, incubation solutions were changed to ATP depletion buffer followed by ATP repletion solution as described in EXPERIMENTAL PROCEDURES. The symbol -//- indicates 20 min of continuous incubation without fluorescence measurements to reduce photooxidative damage. Representative tracing up to 30 min after ATP repletion from 1 of 6 experiments is shown. A, bottom: effect of BAPTA-AM on intracellular calcium after 1 h of ATP depletion and subsequent ATP repletion for 1 h. At indicated time points, BAPTA-AM (50 µM) was added to the ATP depletion buffer for 30 min until it replaced the ATP repletion solution. Representative tracing up to 30 min after ATP repletion from 1 of 5 experiments is shown. B: compiled intracellular calcium levels measured from 5 independent experiments. From left to right: column 1, control MDCK monolayer; column 2, ATP depletion for 1 h; column 3, 1-h ATP depletion followed by 1-h ATP repletion; column 4, ATP depletion for 1 h; column 5, 1-h ATP depletion in the presence of BAPTA-AM; column 6, 1-h ATP depletion followed by 1-h ATP repletion in the presence of BAPTA-AM.

Calcium chelation retards occludin redistribution following ATP depletion-repletion. Because TER is a measure of TJ function and the recovery of TER and paracellular permeability were found to be inhibited by intracellular calcium chelation following ATP depletion-repletion, we examined whether lowered intracellular calcium had an effect on the cellular distribution of two well-characterized TJ proteins, ZO-1 and occludin. As shown in Fig. 4, immunofluorescence microscopy analysis demonstrated that a substantial fraction of occludin, the membrane TJ protein, took on an intracellular distribution after ATP depletion (Fig. 4f). After ATP repletion, occludin was once again found almost exclusively at the level of the TJ (Fig. 4g). This reversible shift was inhibited by the chelation of intracellular calcium (Fig. 4h), although there were subtle differences in the staining pattern between the ATP-depleted cells and the cells subjected to ATP depletion-repletion in the presence of BAPTA-AM. In contrast, the staining pattern of ZO-1 was not significantly altered during ATP depletion or repletion, as we have previously observed (Fig. 4, b and c; Ref. 45), nor was ZO-1 staining affected by treatment with BAPTA-AM (Fig. 4d). Our findings suggest that in this model system for ischemic injury intracellular calcium plays a role in resorting of occludin to the plasma membrane.


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Fig. 4.   Intracellular calcium chelation retards occludin redistribution following ATP depletion-repletion. MDCK cells grown on glass coverslips were fixed and processed for immunostaining using specific antibodies [a-d: zonula occludens (ZO)-1; e-h: occludin] and then analyzed by immunofluorescence microscopy as described in experimental procedures. Data are representative of 5 experiments. a and e: Control monolayers. b and f: ATP depletion for 1 h. c and g: ATP depletion for 1 h and repletion for 3 h. d and h: and ATP depletion-repletion in the presence of BAPTA-AM (50 µM).

Calcium chelation prevents ZO-1 and to a lesser extent ZO-2 and occludin from returning to the soluble pool after ATP repletion. Given that calcium chelation had an effect on the recovery of TER, paracellular flux, and the immunostaining pattern of occludin after ATP repletion, we examined whether there were any correlated alterations in the molecular association between TJ proteins and the actin-based cytoskeleton by using the Triton X-100 extractability assay, a biochemical approach to analyze interactions between junctional proteins and the cytoskeleton (9, 32, 42, 45). As shown in Fig. 5, Triton X-100 solubility of ZO-1 and ZO-2, as well as occludin, decreased (i.e., became less extractable) after ATP depletion, and the changes were reversed after ATP repletion. However, this reversible shift in solubility was inhibited by BAPTA-AM in a dose-dependent manner for ZO-1 and to a lesser degree for ZO-2 and occludin (Fig. 5). Interestingly, the higher-molecular-weight form of the Triton X-100-insoluble occludin, which represents the highly phosphorylated form (36), was not significantly altered by the treatment with BAPTA-AM, although there was an increase in abundance after ATP repletion (Fig. 5). These differential effects of BAPTA-AM on the Triton X-100 extractability of TJ proteins support a functional role for intracellular calcium in the interactions between TJ proteins and the cytoskeleton, especially during the reassembly of the TJ that follows ATP repletion.


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Fig. 5.   Treatment with BAPTA-AM during ATP depletion prevents tight junction (TJ) proteins, particularly ZO-1, from returning to the soluble pool after ATP repletion. MDCK cell monolayers were extracted with CSK-1 buffer containing 0.5% Triton X-100 and 100 mM NaCl in buffered Tris (pH 7.4) for 20 min at 4°C with gentle agitation. Both extractable (E) and residue (R) fractions were separated by 7.5% SDS-PAGE and transferred to nitrocellulose filters and probed with the indicated antibodies. C, control monolayer; D, 1-h ATP depletion; R, 1-h ATP depletion and 3-h ATP repletion; B25, 1-h ATP depletion and 3-h ATP repletion with BAPTA-AM (25 µM); B50, 1-h ATP depletion and 3-h ATP repletion with BAPTA-AM (50 µM). Data are representative of 3 experiments.

Large macromolecular complexes containing ZO-1 and occludin remain after ATP repletion in the BAPTA-AM-treated cells. Using rate zonal centrifugation analysis of the detergent-soluble fraction, we have previously observed an increase in the amount of ZO-1 in high-density fractions following ATP depletion, reflecting the formation of large complexes (45). Because of the effects of BAPTA-AM on TJ protein solubility and localization, we examined whether intracellular calcium had a functional role in stabilizing these protein complexes. As shown in Fig. 6A, the amount of detergent-extractable ZO-1 present in the higher sucrose density fractions increased following ATP depletion and normalized following ATP repletion in a pattern similar to that for the control cells. However, in the BAPTA-AM-treated cells, this reversibility was inhibited, as the complexes containing ZO-1 remained in the high-density fractions. A similar effect was seen with occludin; there was an increase in the amount of occludin found in the higher-density fractions on treatment with BAPTA-AM (Fig. 6B). These results suggest that intracellular calcium may be required not only in the interactions between TJ proteins and the cytoskeleton, as suggested by the Triton X-100 extractability assay of the detergent-insoluble fractions (Fig. 5), but also in the stabilization of macromolecular complexes in the detergent-soluble fractions (Fig. 6).


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Fig. 6.   Macromolecular complexes formed after ATP depletion are stabilized after ATP repletion by chelation of intracellular calcium. MDCK cell monolayers treated as indicated were extracted with a buffer containing 1% Triton X-100, 0.5% deoxycholate, and 0.2% SDS, as defined in experimental procedures. Extracts were layered on top of 5-20% sucrose gradients and centrifuged at 32,000 rpm for 24 h at 4°C. Fractionated samples [from fraction 1 (5% sucrose) to fraction 16 (20% sucrose) and the pellet, fraction 17] were separated on 7.5% SDS-PAGE, transferred to nitrocellulose filters, and probed with ZO-1 antibody (A) or occludin antibody (B). Gradients were standardized with molecular markers as indicated. Data are representative of 3 separate experiments.

To further characterize these large TJ protein-containing complexes formed after ATP depletion and stabilized by lowered intracellular calcium, immunoprecipitations of the detergent-soluble pool of metabolically labeled cell lysates were performed with antibodies against ZO-1 and occludin. We observed a similar pattern between the BAPTA-AM-treated cells and untreated cells with no consistent additional bands for both ZO-1 and occludin immunoprecipitations (data not shown). This suggests that the high-molecular-weight TJ protein-containing complexes identified on sucrose gradient analyses of the detergent-soluble pool may be a consequence of increased self-association of TJ proteins rather than association with other high-molecular-weight proteins.

Calcium chelation stabilizes ZO-1-fodrin interaction after ATP depletion-repletion. We have previously reported that ATP depletion results in an interaction (direct or indirect) between ZO-1 and fodrin (45), and others have also reported that ZO-1 interacts with fodrin under certain conditions (3, 4, 19). Because calcium chelation prevented ZO-1 from returning to the soluble pool after ATP repletion (Fig. 5), we decided to investigate whether it had an effect on the interaction between ZO-1 and fodrin-like cytoskeletal proteins. Immunoprecipitations with anti-ZO-1 antibodies followed by Western blot and probing with anti-fodrin antibody were then carried out. In our previous study, we used a high ionic strength (1 M NaCl) buffer (CSK-2) as a second step to enrich a subfraction of ZO-1 following extraction by CSK-1, which has low ionic strength (100 mM NaCl) (45). We demonstrated the association between this subfraction of ZO-1 and fodrin after ATP depletion. In this study, we improved this procedure by using a one-step buffer (1% Triton X-100, 1% NP-40, 1.5 mM MgCl2, and 150 mM NaCl), which has a higher detergent concentration yet physiological ionic strength (44). By using this buffer, we no longer need to use the high-salt, two-step extraction but have sufficient yield of ZO-1 to analyze its association with fodrin. Under this condition, we observed an increased association of ZO-1 with fodrin in the BAPTA-AM-treated cells compared with the untreated cells following ATP repletion (Fig. 7); this apparently enhanced cytoskeletal interaction may in part account for the decreased extractability of ZO-1 with BAPTA-AM treatment.


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Fig. 7.   BAPTA-AM treatment during ATP depletion enhances an association of ZO-1-containing complex with a cytoskeletal protein, fodrin, after ATP repletion. MDCK cell monolayers were scraped into a buffer containing 1% Triton X-100, 1% NP-40, 150 mM NaCl, 20 mM HEPES (pH 7.5), 300 mM sucrose, 1.5 mM MgCl2, and 1 mM EGTA and extracted for 30 min at 4°C. Supernatant was separated by centrifugation and subjected to immunoprecipitation with anti-ZO-1 antibody. Immunocomplexes were separated by 6% SDS-PAGE and transferred to a nitrocellulose filter. Filter was sequentially probed with anti-fodrin, anti-ZO-1, or anti-ZO-2 antibodies as indicated. R, 1-h ATP depletion and 3-h ATP repletion; B, 1-h ATP depletion and 3-h ATP repletion with treatment with BAPTA-AM (50 µM); N, rat nonimmune serum.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the important role of extracellular calcium in the development of the junctional complex is well established, recent studies have indicated that intracellular calcium is also critical for TJ biogenesis, at least in the calcium switch model (33, 40, 43). In this model, global and local (at the regions of cell contact) increases in intracellular calcium occur during junction formation (33). When these changes in intracellular calcium were buffered by chelation, a disruption of TJ assembly was observed, as indicated by attenuated TER development, altered sorting of ZO-1, as well as altered association between the TJ and the cytoskeleton; however, in this model, unlike ATP depletion-repletion, calcium chelation diminishes the TJ-cytoskeletal interaction (43). In addition to absolute levels of intracellular calcium, thapsigargin-sensitive calcium stores have also been found to be critical for the development of the TJ in the calcium switch model (40). Based on these and others findings, the existence of classic signaling pathways involving calcium in the assembly and reassembly of the TJ has been hypothesized (6). The recent emergence of the ATP depletion-repletion model in MDCK cells for epithelial ischemia has allowed us to examine how the TJ reassembles in the context of recovery from epithelial injury (27, 28, 45, 45a). In this model, we observed that ATP depletion leads to a disruption of the TJ barrier and is associated with an interaction between TJ proteins and the submembranous actin cytoskeleton, as well as formation of large ZO-1-containing complexes, but without a clearly detectable change in the localization of ZO-1. This is roughly the opposite of what happens during TJ disassembly in the calcium switch, which is a much more widely utilized but less pathophysiologically relevant model (6). Because these changes are reversible after ATP repletion, the ATP depletion-repletion model provides a useful system to examine how the TJ barrier is reestablished after injury and to analyze possible mechanisms or factors that are critical for the reassembly of the TJ. We have previously shown that TJ reassembly in this model involves the activation of a tyrosine kinase (45a). In the present study, we show that lowering the intracellular calcium level has a significant effect on TJ recovery after ATP depletion-repletion, which is evidenced by marked inhibition of functional recovery of the permeability barrier as measured by both TER and mannitol flux (Figs. 1 and 2). Because ATP levels in calcium-chelated and noncalcium chelated cells are not significantly different after 3 h of ATP repletion (P >=  0.4), this argues against the possibility that the functional, morphological, and biochemical changes observed in BAPTA-treated cells are due to delayed ATP recovery and suggests that there are other mechanisms for inhibiting the process of TJ repair, which are likely to be calcium mediated. Functional recovery of the TJ after ATP repletion requires proper reassembly of the TJ complex at the cell border and remodeling the architecture of the subplasmalemmal cytoskeleton, both of which seem to be dependent on intracellular calcium. The role of intracellular calcium in these processes could be permissive or instructive or both; at the very least, our studies indicate an important permissive role for this ion.

Recovery of TJ function after ATP repletion requires proper sorting of the TJ components. By immunostaining, we observed that there is an increased intracellular distribution of occludin after ATP depletion-repletion in the presence of BAPTA-AM, suggesting that resorting of this TJ protein involves a calcium-dependent step. In contrast, staining for ZO-1 was not affected significantly by either ATP depletion or calcium chelation (Fig. 4), whereas calcium chelation appears to prevent ZO-1 from returning to the soluble pool after ATP repletion (Fig. 5). Immunofluorescent analysis and detergent extractability represent different approaches for detecting changes in the behavior of TJ proteins under various conditions. Many factors could potentially account for the discrepancy between these two approaches. For example, in this case, we propose that those large complexes containing ZO-1 are formed and become more insoluble (more associated with the cytoskeleton, and therefore, less extractable) in the BAPTA-AM-treated cells and yet remain at or close to the TJ and do not move intracellularly. Therefore, these changes will not be readily detected by immunofluorescence. The retention of ZO-1 at the region of the TJ may also reflect a requirement for this molecule at the site of the injured TJ in order for reassembly of the entire structure to occur after ATP depletion-repletion. In this context, it is interesting to note that in the calcium switch model there are marked changes in the distribution of ZO-1, and its resorting appears to be affected by calcium chelation (43).

To assess the intermediate steps in TJ reassembly, we employed rate zonal ultracentrifugation to analyze the soluble pool following ATP depletion-repletion and the effect of BAPTA treatment. Consistent with our previous findings, large macromolecular complexes containing ZO-1 formed after ATP depletion and resolved following ATP repletion (Fig. 6). These large complexes, incidentally, are not detectable on similar gradients during TJ disassembly following the calcium switch (42, 43). Interestingly, the large complexes containing ZO-1 and occludin continued to be present after ATP repletion in the BAPTA-treated cells (Fig. 6). In this detergent-soluble pool, immunoprecipitations of metabolically labeled cells suggested that ZO-1 and occludin may be self-associated or, in the case of ZO-1, complexes of ZO-1, ZO-2, and ZO-3 may be stochiometrically self-associated.

In polarized epithelial cells, much of the actin is localized under the apical junctional complex along with myosin II and several actin binding proteins, thereby forming the perijunctional ring, which projects onto the cytoplasmic surface of the TJ (18). It is known that actin-disrupting drugs, such as cytochalasin, perturb the paracellular barrier (38). Among the TJ proteins on the cytoplasmic surface, ZO-1 has been proposed to interact with the actin-cytoskeleton through spectrin (3, 4). We have recently shown that fodrin, a nonerythroid spectrin, concentrated in the TJ after ATP depletion and ZO-1 became more tightly associated with fodrin following ATP depletion in MDCK monolayers (45). Here, we show that the interaction between ZO-1 and the cytoskeleton during TJ reassembly can be modulated by intracellular calcium, as evidenced by the detergent salt extraction assay (Fig. 5) and, more specifically, by an altered association between ZO-1 and fodrin after calcium chelation (Fig. 7).

It is interesting to note that the overall level of intracellular calcium remained stable during the period of ATP depletion and repletion in MDCK cells, although the possibility of localized changes of calcium cannot be excluded (Fig. 3). Others have also observed that there is no significant increase of total intracellular calcium within 1 h following ATP depletion alone (without calcium ionophore) in MDCK cells (37, 46), although in other systems, increases of intracellular calcium up to 1 mM have been reported (48). The injurious role of intracellular calcium in epithelial tissue injury has been debated for more than a decade. Many have argued that intracellular calcium levels should be lowered to attenuate epithelial injury. Our results, which suggest that lowering intracellular calcium prevents reestablishment of the permeability barrier, may thus be viewed as somewhat surprising. Most likely this reflects multiple roles for intracellular calcium in different steps of the injury and repair processes. Thus certain processes leading to cell death may be activated by calcium, but, after sublethal injury of the kind elicited by short-term ATP depletion, other processes necessary for recovery, such as the reassembly of TJs, require adequate levels of intracellular calcium. We propose that intracellular calcium is important for the reassembly of a functional TJ following ATP depletion-repletion. In particular, calcium appears to modulate the dissociation of large, insoluble macromolecular complexes between the TJ and cytoskeletal proteins (e.g., fodrin), which accumulate after ATP depletion. This role could be direct or indirect, perhaps through the regulation of a calcium-dependent kinase.


    ACKNOWLEDGEMENTS

We thank the members of Dr. S. K. Nigam's lab for discussions and critical reading of this manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-53507 (to S. K. Nigam) and was done during the tenure of an Established Investigatorship (to S. K. Nigam) of the American Heart Association. J. Ye was supported by National Research Service Award 1 F32 DK-09683-01 (from NIDDK). T. Tsukamoto was partially supported by Uehara Memorial Foundation (Japan). A. Sun was supported by NIDDK Grant DK-47403 and a supplemental grant from the American Society of Nephrology.

Present address of S. K. Nigam and address for reprint requests and other correspondence: Depts. of Pediatrics and Medicine, Div. of Nephrology and Hypertension (0693), 9500 Gilman Dr., Univ. of California-San Diego, La Jolla, CA 92093.

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

Received 4 November 1998; accepted in final form 18 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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