1Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, and 2Division of Surgery, Children's Hospital, Boston, Massachusetts 02115
Submitted 25 November 2002 ; accepted in final form 8 July 2003
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ABSTRACT |
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G proteins; tight junction; adherens junction; Rho
The tight junction is the most apical component of the junctional complex, which includes gap junctions, adherens junctions, and tight junctions. The tight junction separates apical and basolateral membrane domains, a feature essential for cellular polarity, and also regulates paracellular flux through the interaction of transmembrane proteins (claudin and occludin families). Understanding mechanisms regulating maintenance and assembly of junctions is essential for insights into developmental processes and pathophysiological disorders, including malignant transformation and ischemic/toxic injuries. Multiple signaling pathways, including regulated calcium stores, protein kinase C, Src tyrosine kinases, small G proteins, and heterotrimeric G subunits, have been shown to regulate this complex process (for review see Refs. 9 and 39).
The G12 protein family consists of the ubiquitously expressed members G12 and G
13 (54), which regulate a variety of cellular responses, including transformation of fibroblasts (23, 63, 66), activation of JNK and serum response element (7, 15, 32, 46), stress fiber formation (3), and neurite retraction in PC12 cells (27). In addition, G
12 and G
13 can stimulate phospholipase D and transcription of cyclooxygenase-2 and Egr-1 (44, 53, 64). Utilizing inducible G
12 wild-type (wt
12)- and constitutively active Q229L
12 (QL
12)- expressing MDCK cell lines, we demonstrate that QL
12 reversibly disrupts tight and adherens junction proteins, alters polarized localization of membrane proteins, and increases paracellular permeability through activation of Src tyrosine kinase pathways.
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MATERIALS AND METHODS |
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Cell culture. Tet-Off MDCK cells were incubated at 37°Cin 5% CO2 and maintained in Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) containing 5% Tet system-approved FBS (Clontech) and 100 µg/ml G418, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Sigma, St. Louis, MO). MDCK clones were maintained in medium containing 100 µg/ml hygromycin, 100 µg/ml G418, and 40 ng/ml doxycycline. Subsequent experiments with conditional expression of the G subunits were carried out in the same medium without hygromycin and G418 and with or without doxycycline.
Dome formation and inhibitor studies. G12- and QL
12-transfected Tet-Off MDCK cells were grown to confluence in 12-well plates with doxycycline (40 ng/ml) before they were switched to medium with or without doxycycline and with or without the tyrosine kinase inhibitors genistein (100 µM) or PP-2 (10 µM). Dome formation was evaluated by counting typical dome structures with an inverted microscope (Eclipse TE300, Nikon) at x100 magnification in 10 consecutive visual fields.
Immunohistochemistry. G12- and QL
12-transfected Tet-Off MDCK cells were plated on glass coverslips or Transwell filters and grown to confluency with or without doxycycline for specific times. The cells were fixed with 4% paraformaldehyde (EM Sciences, Fort Washington, PA) in PBS for 20 min at room temperature or 100% methanol at -20°C and rinsed with PBS three times for 5 min. Immunohistochemistry was performed in various double-staining combinations. The cells were blocked for 45 min at room temperature in 5% (wt/vol) nonfat milk. Primary antibody combinations were diluted 1:100 in PBS or ZO-1 hybridoma medium and incubated at 4°C for 1.5 h with periodic gentle shaking. Three rinses with 0.05% Triton X-100 in PBS for 5 min each at room temperature preceded the 1-h incubation at 4°C with the corresponding secondary antibody mix. Texas red or fluorescein isothiocyanate-conjugated secondary antibodies (Pierce, Rockford, IL) were diluted 1:100 in 0.05% Triton X-100 in PBS. Coverslips were washed three times with 0.05% Triton X-100 in PBS for 5 min at room temperature, mounted with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL), and viewed through a x100 oil immersion objective with a laser scanning confocal system (model MRC-1024/2p, Bio-Rad) coupled to a Zeiss Axiovert microscope. Localization of proteins was determined after separate excitation/emission of both labeled proteins. Images were processed using Photoshop software (Adobe).
Immunoprecipitation and Western immunoblot analysis. The wt12- or QL
12-expressing cells were cultured with or without doxycycline for 3 days. To obtain whole cell lysates, monolayers were scraped in lysate HEPES buffer (100 mM NaCl, 2 mM EDTA, 10 mM HEPES, pH 7.5, 1 mM NaVO4,25 mM NaF, 1 mM PMSF, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) and subjected to brief sonication on ice. Protein A-Sepharose beads and antibodies against
-catenin,
-catenin, ZO-1, occludin, and claudin-1 were added to the lysate overnight at 4°C on a shaker. Beads were washed four times with lysate buffer, and proteins were eluted in SDS-PAGE sample buffer. SDS-polyacrylamide gelelectrophoresed proteins were transferred overnight (nitrocellulose; MSI, Westboro, MA). Nonspecific binding was blocked with 2% nonfat milk or 2% BSA in blocking buffer (50 mM Tris·HCl, 10 mM EDTA, and 1% Triton X-100) before exposure of the membrane to the primary antibody for 1 h at room temperature. After the lysates were washed (1 h in Tris-buffered saline + 0.05% Tween 20; Sigma), secondary horseradish peroxidase-conjugated antibodies (Jackson Laboratories, West Grove, PA) were used at 1:10,000 for 1 h at room temperature, and signal was detected with SuperSignal West Pico horseradish peroxidase substrate system (Pierce) and autoradiography (Biomax MR, Eastman Kodak, Rochester, NY).
Transepithelial resistance measurements. MDCK cells were plated on polycarbonate filters (Transwell, Costar) at 3 x 105 cells/cm2 and maintained in culture medium + doxycycline for 48-72 h to establish tight monolayers. Similar effects on transepithelial resistance (TER) were obtained when cells were plated at 1.2 x 106 cells/cm2. TER was measured at different times after change to a doxycycline-free medium with a Millipore electrical resistance system, as described previously (55). Measurements (in ohm·cm2) are expressed as a mean of the original readings after subtraction of background values.
Mannitol flux rates. MDCK cells expressing wt12 and QL
12 were cultured for 3 days on 6.5-mm Transwell filters in the presence of doxycycline before half of the filters were switched to doxycycline-free medium with or without tyrosine kinase inhibitors for an additional 3 days. Filters were washed three times with Hanks' balanced salt solution (HBSS) and incubated in HBSS with 1 mM mannitol in both chambers. At time 0, 4 µCi of [3H]mannitol (17 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) were added to the apical chamber, and plates were stirred using an orbital shaker at 37°C. Aliquots (100 µl) were removed from the basal chamber at 30, 60, 120, and 180 min and replaced with an identical volume of HBSS + 1 mM mannitol. Counts were determined in scintillation fluid using a liquid scintillation counter (Packard Tri-Carb A2200). Background was subtracted, counts per minute were converted to picomoles of mannitol, and rates were determined from linear regression analysis of the four time points using GraphPad Prism.
Rho activation assay. After 3 days of culture with or without doxycycline and tyrosine kinase inhibitors, wt12- or QL
12-expressing cells were lysed with ice-cold lysis buffer [50 mM Tris·HCl, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, and protease inhibitors as supplied by the manufacturer (Upstate Biotechnology)]. Samples were sonicated on ice and centrifuged for 10 min at 13,000 rpm. Four hundred microliters of supernatant were added to 30 µl of glutathione-agarose slurry linked to rhotekin Rho-binding domain, which specifically binds activated (GTP-bound) Rho (buffer: 50 mM Tris·HCl, pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and protease inhibitors), and incubated for 45 min at 4°C. Agarose beads were centrifuged and washed three times. Activated Rho was eluted with SDS-PAGE sample buffer, denatured for 5 min at 95°C, and analyzed by Western blot with purified polyclonal anti-RhoA, anti-RhoB, or anti-RhoC antibody (Upstate Biotechnology). Positive and negative controls were prepared with uninduced wt
12- or QL
12-expressing cell lysates. Before incubation with glutathione-agarose slurry, guanosine 5'-O-(3-thiotriphosphate) (100 µM final concentration) or GDP (1 mM final concentration) was added to 400 µl of supernatant at 30°C for 15 min.
Statistics. Values are means ± SE; n refers to the number of measurements. Paired Student's t-test was used to compare mean values within one experimental series. Data from two groups were compared by unpaired t-test. P < 0.05 was accepted to indicate statistical significance.
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RESULTS |
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Expression of QL12 in MDCK cells disrupts tight and adherens junction proteins. We next examined the staining pattern of junctional proteins in the presence and absence of wt
12 and QL
12 expression (Fig. 2). We previously showed that the pattern of ZO-1 staining was significantly altered with QL
12 expression, and we now extend these observations to other tight and adherens junction proteins. There were no observable differences in staining patterns for tight and adherens junction proteins with induced wt
12 expression (results not shown), and there were no differences between cells grown on coverslips and those grown on Transwell filters. In QL
12-expressing MDCK cells, the tight junction proteins claudin-1 and occludin and the adherens junction proteins E-cadherin and
-catenin show altered localization. The patterns were similar for ZO-1, occludin,
-catenin, and E-cadherin, with thickened and disrupted linear membrane staining (Fig. 2). The localization of claudin-1 was most perturbed, with nearly complete loss of membrane staining and development of punctate intracellular staining (Fig. 2, C and D). Activation of
-catenin often results in translocation into the cytoplasm and to the nucleus (36). In QL
12-expressing MDCK cells,
-catenin tyrosine phosphorylation was increased (see Fig. 5), but we did not detect significant cytosolic localization of
-catenin or any colocalization with the nuclear stain 4',6-diamidino-2-phenylindole (not shown).
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Epithelial cell polarity is disrupted and cortical actin staining increased with QL12 expression. Because expression of QL
12, but not wt
12, resulted in significant changes in junctional protein staining and increased paracellular permeability, polarity of the epithelial cell monolayer was likely to be disrupted. The tight junction scaffolding protein ZO-1 is normally restricted to a discrete plane in the tight junction at the interface between the apical and the basolateral membrane domains. Na-K-ATPase is more broadly expressed along the basolateral membrane but restricted from the apical membrane domain. Normally, there is little overlap of these two proteins in MDCK cells, and in the presence of doxycycline, this separation is apparent in QL
12-expressing MDCK cells (Fig. 3A, a and e, and c and g). However, induction of QL
12 protein (doxycycline-free medium) caused loss of this separation, with ZO-1 appearing diffusely along the lateral membrane and in the apical membrane (Fig. 3A, f, and Fig. 3B, b, arrow) and the Na-K-ATPase partially localizing in the apical membrane (Fig. 3A, d, and Fig. 3B, d, arrow). Expression of wt
12 caused no significant changes in the actin cytoskeleton (see Fig. 6A), whereas induction of QL
12 protein resulted in altered cell shape and increased actin stress fibers (Fig. 3B, e and f; see Fig. 6B). Actin staining was similar in QL
12- and wt
12-expressing MDCK cells in the absence of G
12 expression (doxycycline-containing medium; see Fig. 6). These findings indicate that QL
12 expression causes cell rounding, increased stress fibers, and loss of separation between proteins that are normally restricted to specific membrane domains.
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Paracellular regulation of MDCK cell junctions by G12 involves Src tyrosine kinase(s). Tyrosine kinases have been shown to regulate epithelial cell junctions (56, 61), and several have been shown to be immediate downstream effectors of G
12 (24). We found that genistein (a well-described broad inhibitor of tyrosine kinases) and PP-2 [a highly specific Src kinase family inhibitor that has been crystallized with a Src family member (51)] prevented the decrease in TER seen in QL
12-expressing MDCK cells (Fig. 4A). Baseline TER in Tet-Off MDCK cells was
75
·cm2, which is a little lower than values reported by some groups (26) but consistent with values reported by others (34). Neither PP-2 nor genistein affected baseline TER under control conditions (doxycycline-containing medium), and neither compound affected G
12 protein expression or levels of ZO-1 (by Western blot; not shown). PP-2 completely blocked the fall in TER typically seen in QL
12-expressing MDCK cells. Interestingly, genistein caused a significant increase in TER throughout the experiment. The effects of PP-2 and genistein on blocking the QL
12-induced fall in TER were completely reversible. With removal of the inhibitors and return to doxycycline-free medium (at
120 h; Fig. 4A), there was a rapid fall in TER to the level seen in untreated monolayers. To confirm that the effects of the inhibitors were on paracellular flux and not on transcellular ion flow, we measured paracellular flux of [3H]mannitol in wild-type and QL
12-expressing MDCK cells in the presence and absence of G
12 expression and inhibitors. Figure 4B demonstrates a large (
7-fold) increase in paracellular flux with induction of QL
12 expression. The increase in flux from QL
12 expression was significantly but not completely inhibited by PP-2 and genistein. Both inhibitors resulted in a small increase in flux of monolayers in doxycycline-containing medium, perhaps because of nonspecific effects from exposure to the inhibitors for 72 h. Although much less dramatic, similar findings were observed in wt
12-overexpressing MDCK cells. There was a small increase in mannitol flux that was also blocked by the inhibitors (Fig. 4B).
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Src is activated and -catenin phosphorylation increased without detectable Rho activation in QL
12-expressing MDCK cells. On the basis of the observation that PP-2 blocked the QL
12 protein effects on the MDCK cell junction and the report that Src is activated by G
12 (28, 41), we investigated c-Src activity in QL
12- and wt
12-expressing cells. Western blots of wt
12- and QL
12-expressing MDCK cell lysates prepared from cells grown in medium with and without doxycycline showed no significant difference in total c-Src immunoreactivity (Fig. 5A). Src is activated by phosphorylation of pTyr419 but is normally maintained in an inactive conformation through intramolecular interactions between the SH2 domain and the phosphorylated COOH-terminal tail (pTyr530) (for review see Ref. 58). Therefore, increases in phosphorylation of Tyr419 or decreases in Tyr530 can be used as evidence of Src activation. Without G
12 expression (doxycycline-containing medium), phosphorylation of pTyr419 (as detected with pTyr419-specific antibody) was minimal in wt
12- and QL
12-expressing MDCK cells, reflecting Src inactivation (Fig. 5A, middle). Inducing QL
12 expression (doxycycline-free medium) caused a significant increase of pTyr419 phosphorylation (4.4 ± 0.4 fold, n = 5) with no detectable change in wt
12-expressing MDCK cells (Fig. 5A, middle). Also, consistent with c-Src activation, there was a concomitant decrease of pTyr530 phosphorylation (3.9 ± 0.3 fold, n = 4) with QL
12 expression, whereas there was no significant difference in pTyr530 Src phosphorylation relative to occludin in wt
12-expressing cells (Fig. 5A, right).
Our confocal studies showed that E-cadherin and -catenin localization were disrupted in QL
12-expressing MDCK cells (Fig. 2). G
12 recently was demonstrated to interact with E-cadherin and regulate
-catenin release (35), and other studies have shown that
-catenin tyrosine phosphorylation is increased during adherens junction disassembly (72). To determine whether QL
12 expression caused increased phosphorylation of
-catenin, we immunoprecipitated
- and
-catenin from QL
12-expressing MDCK cells in medium with and without doxycycline. In our gel system,
- and
-catenin migrate with nearly identical molecular mass of 98 kDa. Nevertheless, Fig. 5B (top) shows that
-catenin precipitates a significant amount of
-catenin, whereas the
-catenin coprecipitates a much smaller amount of
-catenin. Both
- and
-catenin were immunoprecipitated from cells in medium with and without doxycycline and probed by Western blot with anti-pTyr antibodies (Fig. 5B, bottom). For
-and
-catenin precipitates, there is a significant increase in tyrosine phosphorylation of cells in medium without doxycycline. This finding, combined with the results of Fig. 5B, top, suggests that most tyrosine phosphorylation is occurring on
-catenin, although we cannot rule out changes in
-catenin as well. Similar results were obtained when filters were stripped and reprobed with catenin or pTyr antibodies.
Because G12 can activate Rho through Src-dependent and Src-independent mechanisms, we determined whether Rho was activated with QL
12 expression. G
12 regulation of the actin cytoskeleton is directly linked to Rho signaling via a novel family of Rho exchange proteins [p115 RhoGEF (29)], and Rho has been shown to regulate tight junctions (25, 26, 47, 65). However, in rhotekin-binding assays (Fig. 5C), the amount of activated Rho was not increased in wt
12-or QL
12-expressing MDCK cells 72 h after removal of doxycycline. There was no detectable difference in the quantity of Rho in cell lysates, nor did treatment with PP-2 or genistein significantly affect Rho expression or activation. In support of this observation, treatment of G
12-expressing MDCK cells with the cell-permeant Rho kinase inhibitor Y-27632 had no effect on QL
12-induced changes in the junction (results not shown).
Tyrosine kinase inhibitors prevent disruption of junctional proteins in QL12-expressing MDCK cells. Confocal analysis of QL
12-expressing MDCK cells reveals that expression of wt
12 has little effect on the pattern of ZO-1 localization and actin staining at the level of the tight junction (Fig. 6A). Similar to our previous report (37), wt
12 was found along the cell perimeter (Fig. 6A, d), and treatment with genistein or PP-2 did not appear to significantly change the G
12 staining pattern. Actin staining was unchanged by wt
12 expression (Fig. 6A, c and f) and was not significantly affected by the inhibitors, although a few more stress fibers were evident (Fig. 6A, i and l). In QL
12-expressing MDCK cells, the inhibitors had no major effect on QL
12 localization, but in QL
12-expressing MDCK cells treated with PP-2 or genistein, the inhibitors prevented the disrupted ZO-1 staining pattern normally observed in MDCK cells expressing QL
12 (Fig. 6B, b, e, h, and k). In PP-2 treated cells, the ZO-1 staining pattern was indistinguishable from controls, with a fine linear continuous staining pattern along the lateral membrane (Fig. 6B, k and b). Interestingly, genistein had a novel effect on cell morphology. The ZO-1 staining remained narrow and continuous, but the membrane was no longer linear and contained "microspikes" (Fig. 6B, h). This effect of genistein on cell morphology was not seen in wt
12-expressing MDCK cells (Fig. 6A, h). Unlike wt
12-expressing MDCK cells, actin staining of QL
12-expressing MDCK cells revealed increased stress fibers (Fig. 6B, f), and the nature of these stress fibers was different in the presence of the inhibitors. Transcelluar actin filaments appeared significantly shorter in genistein-treated than in untreated cells (Fig. 6B, f and i), whereas PP-2 appeared to increase the number and length of stress fibers. Although the inhibitors caused some changes in morphology, the effect on the barrier was less pronounced. PP-2 had no significant effect on the time course of TER development, and genistein caused a significant increase in TER (Fig. 4A). Furthermore, the effect of the inhibitors on the actin cytoskeleton at the level of the tight junction was less pronounced. Figure 7 shows the actin cytoskeleton at the level of the tight junction protein ZO-1. In doxycycline-containing medium, very few stress fibers are visible at the level of ZO-1, and actin staining is predominantly along the lateral margins of the cell, with only focal areas of overlap (yellow in the merged images). With induction of QL
12 (doxycycline-free medium), ZO-1 staining becomes fragmented and more diffuse along the lateral membrane (Figs. 2 and 6), and most of the ZO-1 is colocalizing with actin. Stress fibers are more abundant, and there is increased intensity of cortical actin staining. In the uninduced cells, ZO-1 staining is preserved, although in genistein-treated cells, stress fibers are more visible and the increased intensity of cortical actin staining is still evident. PP-2-treated cells appear more similar to the control condition, with fewer stress fibers and less intense actin staining. In the presence of inhibitors and doxycycline, the pattern is similar to that in medium containing doxycycline only.
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DISCUSSION |
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Expression of QL12 results in a leaky epithelium (37) and is reflected in these studies by the loss of monolayer domes. Dome formation reflects the tightness of the monolayer and is a sensitive indicator of alterations in junctional integrity (4, 30). The effect of QL
12 on dome formation was reversible, indicating that QL
12 expression did not irreparably injure or alter the cell. The functional changes in paracellular permeability correlated with morphological changes of the junctional complex. With QL
12, but not wt
12, expression, a striking change was observed in the staining pattern of the tight junction proteins ZO-1, occludin, and claudin-1 and the adherens junction proteins E-cadherin and
-catenin. This effect of QL
12 on junctional proteins appears to be unique within the G protein family. Previous studies expressing wild-type or constitutively active mutants of G
o, G
i2, or G
s had no effect on the staining patterns of ZO-1, occludin, or E-cadherin (10, 49, 50). In the QL
12-expressing MDCK cells, claudin-1 localization was most severely affected. Claudins are essential for barrier function (60) and for paracellular regulation of specific ions (52, 62). We hypothesize that the loss of dome formation and barrier function was predominantly mediated by changes in claudin distribution. Because claudins interact with zona occludens proteins (22) and ZO-1 binds to G
12, we speculate that the QL
12-induced changes in ZO-1 localization resulted in the disrupted claudin distribution. However, we cannot exclude the possibility that QL
12 simultaneously disrupts the adherens junction, because tight junction assembly starts with interactions of E-cadherin and E-cadherin also interacts with G
12 (39). Similarly, we found the normal separation of ZO-1 and Na-K-ATPase disrupted in QL
12-expressing MDCK cells. The most likely explanation is the loss of junctional integrity, resulting in diffusion of proteins within the plasma membrane. Although we did not look at other membrane marker proteins and did not explicitly examine protein diffusion, this is most likely a generalized effect of QL
12 on the junction and is not specific to the Na-K-ATPase. This loss of polarity would be expected in epithelial cells with a disrupted junctional complex and changes in protein-protein interactions among junctional proteins.
Several different signaling pathways regulate the barrier of intact epithelia. Activation and overexpression of protein kinase C isoforms or monomeric G proteins, in addition to lowering extracellular calcium, result in disruption of junctions and increased leakiness in kidney epithelial cells (20, 25, 26, 40, 48). Furthermore, we previously showed that activated Gi2 and G
s increase baseline TER in MDCK cells (49, 50). Tyrosine kinases are well described in regulation of epithelial cell junctions and are important downstream effectors of G
12. The tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 prevented development of the typical phenotype in QL
12-expressing MDCK cells. Although the effects of these two inhibitors were different, both prevented the increase in paracellular permeability as measured by the fall in TER and increase in mannitol flux. Both inhibitors prevented the disruption of ZO-1 staining, and genistein treatment resulted in a distinct cell shape and altered actin cytoskeleton, which resulted in a less permeable paracellular space. The effects of genistein compared with PP-2 indicate that, in addition to c-Src, other tyrosine kinase pathways are likely to regulate these processes. We further demonstrated a change in c-Src tyrosine phosphorylation pattern that is consistent with Src activation in QL
12-expressing cells. These studies were not designed to distinguish between direct Src activation by G
12 and indirect activation of Src via another mechanism. G
12 directly regulates other tyrosine kinases [Bruton's tyrosine kinase and pp72syk (24, 28)], and several receptors (thrombin, endothelin, and vasopressin) signal through G
12 to indirectly activate Src (6). Furthermore, direct Src kinase activation by G
12/13 was shown in thrombin-stimulated platelets and coincided with platelet shape changes (28). Although G
12 signaling is linked to Rho-mediated changes in the actin cytoskeleton [via Rho exchange factors (3, 16-18, 28, 29, 31)], no significant Rho activation was detected in these cell lines. We cannot entirely exclude the possibility that transient Rho activation occurred at earlier time points of junction disassembly, but we would suggest that this is unlikely, because the junctional phenotype was dependent on persistent QL
12 expression. It is also possible that other small G proteins (not detected with rhotekin binding), such as Rac1, are downstream of G
12 in this system. In an analogous experimental design, Postma et al. (45) showed that G
12 regulation of gap junctions via Src tyrosine kinases was also independent of Rho activation. The present experiments do not distinguish direct Src phosphorylation of
-catenin from other mechanisms causing increased phosphorylation (e.g., changes in E-cadherin and catenin localization induced by QL
12 expression). Future experiments are needed to determine whether the increased
-catenin phosphorylation is important to the G
12/Src-mediated effects on the junction.
Direct effects of Src on the junctional complex are well established. Tyrosine kinase signaling is important in junctional regulation, and Src, in particular, affects junctional assembly and baseline properties. Activation of Src reduces intercellular adhesion but differentially affects tight and adherens junctions. Oncogenic v-Src induced the redistribution of E-cadherin into the cytosol and caused the disassembly of adherens junctions with only marginally altered tight junction structure and function (19, 56). Avian c-Src expression, which does not cause cell transformation in fibroblasts (57), increased tyrosine phosphorylation of the tight junction proteins ZO-1 and ZO-2 and the adherens junction protein -catenin but not
-catenin (56). This raises the possibility that Src could be directly phosphorylating
-catenin in QL
12-expressing MDCK cells. We previously showed that tyrosine phosphorylation of proteins in the region of the tight junction (possibly by Src) is important for tight junction reassembly after exposure to oxidative stress (38). Similarly, in the ATP depletion-repletion model, tyrosine kinase inhibition prevented recovery of the tight junction after ATP depletion, and tyrosine phosphorylation of occludin was found to be critical for the tight junction reassembly (5, 59).
In recent years, there has been a dramatic increase in the number of proteins found in or near the epithelial cell tight junction. In addition, there are a surprisingly large number of interacting scaffolding proteins of the membrane-associated guanylate kinase (MAGUK) family, now identified within the epithelial cell tight junction. These proteins share a common overall structure and typically include multiple protein-protein interaction domains, including multiple PDZ domains (12, 13). Examples of these proteins, known to be located in the epithelial cell tight junction, include ZO-1, ZO-2, and ZO-3 and the related MAGI-1 (43) proteins. In addition, other interactions between junctional adhesion molecule and another MAGUK protein (CASK/LIN-2) have been identified (33). One question arising from these observations is why the tight junction requires so many interacting scaffolding molecules. We previously demonstrated a direct interaction between G12 and ZO-1 in vitro and in cells and now show that Src tyrosine kinases are important for the G
12-mediated effects on the junction. This raises the possibility that Src is also contained within this multiprotein complex. However, our initial efforts to identify Src in complex with ZO-1 and G
12 have been unsuccessful. This suggests to us that Src is in proximity to ZO-1 and G
12 but may be in direct association with one of the other scaffolding proteins. Our previous results with activated G
i2 and G
s in MDCK cells have shown increased rates of tight junction assembly in the calcium switch model in addition to increased baseline TER. G
12 appears to have the opposite effect on the junction, leading us to speculate that multiple G proteins regulate the properties of the junction in opposing directions. Therefore, one of the reasons for concentrating scaffolding molecules in the tight junction may be to coordinate the spatial and functional interactions of these (and other) signaling proteins. If this hypothesis is correct, then we expect to find more direct interactions between these MAGUK proteins and signaling molecules. The future challenge is to identify these interactions and begin to define these regulatory pathways in vivo.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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