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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
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.
|
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.
|
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.
|
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).
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balda, M. S.,
L. Gonzalez-Mariscal,
K. Matter,
M. Cereijido,
and
J. M. Anderson.
Assembly of the tight junction: the role of diacylglycerol.
J. Cell Biol.
123:
293-302,
1993[Abstract].
2.
Balda, M. S.,
J. A. Whitney,
C. Flores,
G. Sirenia,
M. Cereijido,
and
K. Matter.
Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of mutant tight junction membrane protein.
J. Cell Biol.
134:
1031-1049,
1996[Abstract].
3.
Beck, K. A.,
and
W. J. Nelson.
The spectrin-based membrane skeleton as a membrane protein-sorting machine.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1263-C1270,
1996
4.
Bennett, V.
Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm.
Physiol. Rev.
4:
1029-1065,
1990.
5.
Citi, S.,
H. Sabanay,
R. Jakes,
B. Geiger,
and
J. Kendrick-Jones.
Cingulin, a new peripheral component of tight junctions.
Nature
333:
272-276,
1988[Medline].
6.
Denker, B. M.,
and
S. K. Nigam.
Molecular structure and assembly of the tight junction.
Am. J. Physiol.
274 (Renal Physiol. 43):
F1-F9,
1998
7.
Denker, B. M.,
C. Saha,
S. Khawaja,
and
S. K. Nigam.
Involvement of a heterotrimeric G protein a subunit in tight junction biogenesis.
J. Biol. Chem.
271:
25750-25753,
1996
8.
Drubin, D. G.,
and
W. J. Nelson.
Origins of cell polarity.
Cell
84:
335-344,
1996[Medline].
9.
Fey, E. G.,
K. M. Wan,
and
S. Penman.
Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.
J. Cell Biol.
98:
1973-1984,
1984[Abstract].
10.
Fish, E. M.,
and
B. A. Molitoris.
Alterations in epithelial polarity and the pathogenesis of disease states.
N. Engl. J. Med.
330:
1580-1588,
1994
11.
Furuse, M.,
K. Fujita,
T. Hiiragi,
K. Fujimoto,
and
S. Tsukita.
Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.
J. Cell Biol.
141:
1539-1550,
1998
12.
Furuse, M.,
T. Hirose,
M. Itoh,
A. Nagafuchi,
S. Yonemura,
S. Tsukita,
and
S. Tsukita.
Occludin: a novel integral membrane protein localizing at tight junctions.
J. Cell Biol.
123:
1777-1788,
1993[Abstract].
13.
Furuse, M.,
M. Itoh,
T. Hirase,
A. Nagafuchi,
S. Yonemura,
S. Tsukita,
and
S. Tsukita.
Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.
J. Cell Biol.
127:
1617-1626,
1994[Abstract].
14.
Gopalakrishnan, S.,
N. Raman,
S. J. Atkinson,
and
J. A. Marrs.
Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion.
Am. J. Physiol.
275 (Cell Physiol. 44):
C798-C8099,
1998[Abstract].
15.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of calcium indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
16.
Gumbiner, B.,
T. Lowenkopf,
and
D. Apatira.
Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1.
Proc. Natl. Acad. Sci. USA
88:
3460-3464,
1991[Abstract].
17.
Haskins, J.,
L. Gu,
E. S. Wittchen,
J. Hibbard,
and
B. R. Stevenson.
ZO-3, a novel member of the MAGUK protein family found at the tight junction, interactions with ZO-1 and occludin.
J. Cell Biol.
141:
199-208,
1998
18.
Hirokawa, N.,
and
L. G. Tilney.
Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear.
J. Cell Biol.
95:
249-261,
1982[Abstract].
19.
Itoh, M.,
A. Nagafuchi,
S. Moroi,
and
S. Tsukita.
Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to a catenin and actin filaments.
J. Cell Biol.
138:
181-192,
1997
20.
Itoh, M.,
A. Nagafuchi,
S. Yonemura,
T. Kitani-Yasuda,
S. Tsukita,
and
S. Tsukita.
The 220-kDa protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.
J. Cell Biol.
121:
491-502,
1993[Abstract].
21.
Jesaitis, L. A.,
and
D. A. Goodenough.
Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and Drosophila discs-large tumor suppressor protein.
J. Cell Biol.
124:
949-961,
1994[Abstract].
22.
Jou, T.-S.,
E. E. Schneeberger,
and
W. J. Nelson.
Structural and functional regulation of tight junctions by Rho-A and Rac 1 small GTPases.
J. Cell Biol.
142:
101-115,
1998
23.
Keon, B. H.,
S. Schfer,
C. Kuhn,
C. Grund,
and
W. W. Franke.
Symplekin, a novel type of tight junction plaque protein.
J. Cell Biol.
134:
1003-1018,
1996[Abstract].
24.
Kuznetsov, G.,
K. T. Bush,
P. L. Zhang,
and
S. K. Nigam.
Pertubations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.
Proc. Natl. Acad. Sci. USA
93:
8584-8589,
1996
25.
Kwon, O.,
W. J. Nelson,
R. Sibley,
P. Huie,
J. D. Scanding,
D. Dafoe,
E. Alfrey,
and
B. D. Myers.
Backleak, tight junctions, and cell-cell adhesion in postischemic injury to the renal allograft.
J. Clin. Invest.
101:
2054-2064,
1998
26.
Madara, J. L.
Intestinal absorptive cell tight junctions are linked to cytoskeleton.
Am. J. Physiol.
253 (Cell Physiol. 22):
C171-C175,
1987
27.
Mandel, L. J.,
R. Bacallao,
and
G. Zampighi.
Uncoupling of the molecular "fence" and paracellular "gate" functions in epithelial tight junctions.
Nature
361:
552-555,
1993[Medline].
28.
Mandel, L. J.,
R. B. Doctor,
and
R. Bacallao.
ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K+-ATPase and E-cadherin.
J. Cell Sci.
107:
3315-3324,
1994
29.
McCarthy, K. M.,
I. B. Skare,
M. C. Stankewich,
M. Furuse,
S. Tsukita,
R. A. Rogers,
R. D. Lynch,
and
E. E. Schneeberger.
Occludin is a functional component of the tight junction.
J. Cell Sci.
109:
2287-2298,
1996
30.
Mitic, L. L.,
and
J. M. Anderson.
Molecular architecture of tight junctions.
Annu. Rev. Physiol.
60:
121-142,
1998[Medline].
31.
Molitoris, B. A.,
and
W. J. Nelson.
Alterations in the establishment and maintenance of epithelial cell polarity as a basis for disease processes.
J. Clin. Invest.
85:
3-9,
1990[Medline].
32.
Nelson, W. J.,
and
R. W. Hammerton.
A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity.
J. Cell Biol.
108:
893-902,
1989[Abstract].
33.
Nigam, S. K.,
E. Rodrigues-Boulan,
and
R. B. Silver.
Changes in intracellular calcium during the development of epithelial polarity and junctions.
Proc. Natl. Acad. Sci. USA
89:
6162-6166,
1992[Abstract].
34.
Rodriguez-Boulan, E.,
and
W. J. Nelson.
Morphogenesis of the polarized epithelial cell phenotype.
Science
245:
718-725,
1989[Medline].
35.
Saitou, M.,
K. Fujimoto,
Y. Doi,
M. Itoh,
T. Fujimoto,
M. Furuse,
H. Takano,
T. Noda,
and
S. Tsukita.
Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions.
J. Cell Biol.
141:
397-408,
1998
36.
Sakakibara, A.,
M. Furuse,
M. Saitou,
Y. Ando-Akatsuka,
and
S. Tsukita.
Possible involvement of phosphorylation of occludin in tight junction formation.
J. Cell Biol.
137:
1393-1401,
1997
37.
Sheridan, A. M.,
J. H. Schwartz,
V. M. Kroshan,
A. M. Tercyak,
J. Laraia,
S. Masino,
and
W. Lieberthal.
Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F342-F350,
1993
38.
Stevenson, B. R.,
and
D. A. Begg.
Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells.
J. Cell Sci.
107:
367-375,
1994
39.
Stevenson, B. R.,
J. D. Siliciano,
M. S. Mooseker,
and
D. A. Goodenough.
Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.
J. Cell Biol.
103:
755-766,
1986[Abstract].
40.
Stuart, R. O.,
and
S. K. Nigam.
Development of tubular nephron.
Semin. Nephrol.
15:
315-326,
1995[Medline].
41.
Stuart, R. O.,
and
S. K. Nigam.
Regulated assembly of tight junctions by protein kinase C.
Proc. Natl. Acad. Sci. USA
92:
6072-6076,
1995
42.
Stuart, R. O.,
A. Sun,
K. T. Bush,
and
S. K. Nigam.
Dependence of epithelial intercellular junction biogenesis on tharpsigargin-sensitive intracellular calcium stores.
J. Biol. Chem.
271:
13636-13641,
1996
43.
Stuart, R. O.,
A. Sun,
M. Panichas,
S. C. Hebert,
B. M. Brenner,
and
S. K. Nigam.
Critical role for intracellular calcium in tight junction biogenesis.
J. Cell. Physiol.
159:
423-433,
1994[Medline].
44.
Toshihiko Toyofuku, M. Y.,
K. Otsu,
T. Kuzuya,
M. Hori,
and
M. Tada.
Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.
J. Biol. Chem.
273:
12725-12731,
1998
45.
Tsukamoto, T.,
and
S. K. Nigam.
Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly.
J. Biol. Chem.
272:
16133-16139,
1997
45a.
Tsukamoto, T.,
and
S. K. Nigam.
Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins.
Am. J. Physiol.
276 (Renal Physiol. 45):
F737-F750,
1999
46.
Venkatachalam, M. A.,
J. M. Weinberg,
Y. Patel,
U. Hussong,
and
J. A. Davis.
Effects of Ca2+ and glycine on lipid breakdown and death of ATP-depleted MDCK cells.
Kidney Int.
48:
118-128,
1995[Medline].
47.
Weinberg, J. M.,
J. A. Davis,
N. F. Roeser,
and
M. A. Venkatachalam.
Role of increased cytosolic free calcium in the pathogenesis of rabbit tubule cell injury and protection by glycine or acidosis.
J. Clin. Invest.
87:
581-590,
1991[Medline].
48.
Weinberg, J. M.,
J. A. Davis,
and
M. A. Venkatachalam.
Cytosolic-free calcium increases to greater than 100 micromolar in ATP-depleted proximal tubules.
J. Clin. Invest.
100:
713-722,
1997
49.
Yamamoto, T.,
N. Harada,
K. Kano,
S.-I. Taya,
E. Canani,
Y. Matsuura,
A. Mizoguchi,
C. Ide,
and
K. Kabuchi.
The ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells.
J. Cell Biol.
139:
785-795,
1997
50.
Zhong, Y.,
T. Saitoh,
T. Minase,
N. Sawada,
K. Enomoto,
and
M. Mori.
Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2.
J. Cell Biol.
120:
477-483,
1993[Abstract].