G{alpha}12 regulates epithelial cell junctions through Src tyrosine kinases

Tobias N. Meyer,1 Jennifer Hunt,1 Catherine Schwesinger,2 and Bradley M. Denker1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that G{alpha}12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated G{alpha}12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of G{alpha}12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active G{alpha}12 (QL{alpha}12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QL{alpha}12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QL{alpha}12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QL{alpha}12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QL{alpha}12-expressing MDCK cells as assessed by Src autophosphorylation, and {beta}-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that G{alpha}12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

G proteins; tight junction; adherens junction; Rho


HETEROTRIMERIC G proteins, consisting of G{alpha} and G{beta}{gamma} subunits, are expressed in all eukaryotic cells and provide a major mechanism for signal transduction. Binding of an agonist to a G protein-coupled receptor activates the G{alpha} subunit by promoting GDP release and GTP binding. GTP-liganded G{alpha} dissociates from G{beta}{gamma}, and both subunits interact with downstream effector molecules (for review see Ref. 42). There are >=16 G{alpha} subunits grouped into 4 major families on the basis of sequence similarities of the {alpha}-subunit (Gs, Gi, Gq, and G12), and multiple family members are typically expressed within the same cell. One mechanism to allow specific signal transduction is localization of signaling molecules into discrete membrane domains. Several G{alpha} subunits, including G{alpha}12, partially colocalize within the epithelial cell junction (8, 10, 11, 21). Regulation of the junctional complex by heterotrimeric G proteins was suggested in early experiments (1), and subsequent studies have demonstrated that pertussis toxin-sensitive family members, G{alpha}i2 and G{alpha}o, localize in the tight junction of Madin-Darby canine kidney (MDCK) cells and affect tight junction assembly and baseline properties (8, 10, 21, 50). Recently, the role of G proteins modulating the tight junction has been expanded to include G{alpha}s, which also stimulates tight junction assembly and indirectly associates with a ZO-1 complex (49).

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{alpha} 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 G{alpha}12 and G{alpha}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{alpha}12 and G{alpha}13 can stimulate phospholipase D and transcription of cyclooxygenase-2 and Egr-1 (44, 53, 64). Utilizing inducible G{alpha}12 wild-type (wt{alpha}12)- and constitutively active Q229L{alpha}12 (QL{alpha}12)- expressing MDCK cell lines, we demonstrate that QL{alpha}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials and cell lines. cDNAs for wt{alpha}12 and QL{alpha}12 were kindly provided by Henry Bourne (University of California, San Francisco). Establishment and characterization of Tet-Off MDCK cell lines with inducible G{alpha}12 expression are described elsewhere (37). Polyclonal anti-G{alpha}12 antibodies (S-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-{alpha}- or {beta}-catenin, anti-occludin, anti-claudin-1, and anti-E-cadherin from Zymed Laboratories (San Francisco, CA), monoclonal anti-phosphotyrosine (pTyr; 4G10), monoclonal anti-Na-K-ATPase, and the Rho activation assay from Upstate Biotechnology (Lake Placid, NY), and anti-c-Src, anti-pTyr419-Src, and anti-pTyr530-Src from Biosource International (Camarillo, CA). Anti-ZO-1 rat monoclonal antibody (R40.76) was kindly supplied by D. Goodenough (Harvard University). Phalloidin coupled to Alexa Fluor 568 (excitation at 578 nm and excitation at 600 nm) was purchased from Molecular Probes (Eugene, OR), Tet-Off MDCK type II epithelial (T-23 MDCK) cells, Tet-Off cloning vectors, and tetracycline-free fetal calf serum from Clontech (Palo Alto, CA), genistein from Sigma, PP-2 from Calbiochem (San Diego, CA), plasticware from Falcon (Lincoln Park, NJ), Transwell filters [polycarbonate, permeable (0.4 µm pore, 6 and 12 mm diameter)] from Costar (Cambridge, MA), and the ohm meter (Millicell ERS) from Millipore (Bedford, MA).

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{alpha} subunits were carried out in the same medium without hygromycin and G418 and with or without doxycycline.

Dome formation and inhibitor studies. G{alpha}12- and QL{alpha}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. G{alpha}12- and QL{alpha}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 wt{alpha}12- or QL{alpha}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 {alpha}-catenin, {beta}-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 wt{alpha}12 and QL{alpha}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, wt{alpha}12- or QL{alpha}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{alpha}12- or QL{alpha}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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of activated (QL) G{alpha}12 affects dome formation in MDCK cells. We previously showed that activated G{alpha}12 (QL) expressed in MDCK cells reversibly increased paracellular flux and lowered TER (37). Figure 1 demonstrates another consequence of leaky epithelia, i.e., the loss of domes. Domes are focal areas of fluid accumulation that appear in confluent monolayers of MDCK cells and arise from vectorial transport of fluid across the monolayer. Domes arise in areas where pressure from accumulated fluid exceeds the binding to the culture plate (4, 30), and dome formation can be inhibited by drugs that block transporters or ion pumps (e.g., ouabain) or interventions that disrupt the junctional complex (e.g., low calcium). Figure 1A shows QL{alpha}12 protein levels as a function of doxycycline concentration, and Fig. 1, B and C, shows the inverse correlation between QL{alpha}12 protein levels and dome formation. In the absence of doxycycline, domes were completely absent (Fig. 1B, d), and there was an inverse correlation between QL{alpha}12 protein levels and the number of domes visualized (Fig. 1, A and C). There were no differences among wt{alpha}12- and QL{alpha}12-expressing MDCK cell lines in the presence of doxycycline (no G{alpha}12 expression), and the number of domes was unchanged by inducing wt{alpha}12 protein for 72 h (Fig. 1, B, a and c, and Fig. 1C).



View larger version (56K):
[in this window]
[in a new window]
 
Fig. 1. QL{alpha}12 expression and dome formation in Madin-Darby canine kidney (MDCK) cells. A: Western blot of G{alpha}12 after 48 h of induction of QL{alpha}12-expressing cells with doxycycline (0-40 ng/ml). Expression was resuppressed within 48 h upon readdition of doxycycline after 48 h in the absence of doxycycline (right lane). B: phase-contrast microscopy of wt{alpha}12- and QL{alpha}12-expressing MDCK monolayers at baseline [in doxycycline-containing medium (+dox)] and 72 h after induction of G{alpha}12 expression. Monolayers expressing wt{alpha}12 showed typical dome formation without (a, +dox) or with (c, -dox) induction of wt{alpha}12 expression. In QL{alpha}12-expressing MDCK cells, dome formation was observed without induction of QL{alpha}12 expression (b). After 72 h of QL{alpha}12 expression (d, -dox), dome formation was no longer observed. Scale bar, 50 µm. C: quantitation of dome formation as a function of doxycycline concentration at 72 h of QL{alpha}12 or wt{alpha}12 expression. There is a significant difference in domes of QL{alpha}2 MDCK cells starting at 1 ng/ml doxycycline from uninduced monolayers (40 ng/ml).

 

Expression of QL{alpha}12 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{alpha}12 and QL{alpha}12 expression (Fig. 2). We previously showed that the pattern of ZO-1 staining was significantly altered with QL{alpha}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{alpha}12 expression (results not shown), and there were no differences between cells grown on coverslips and those grown on Transwell filters. In QL{alpha}12-expressing MDCK cells, the tight junction proteins claudin-1 and occludin and the adherens junction proteins E-cadherin and {beta}-catenin show altered localization. The patterns were similar for ZO-1, occludin, {beta}-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 {beta}-catenin often results in translocation into the cytoplasm and to the nucleus (36). In QL{alpha}12-expressing MDCK cells, {beta}-catenin tyrosine phosphorylation was increased (see Fig. 5), but we did not detect significant cytosolic localization of {beta}-catenin or any colocalization with the nuclear stain 4',6-diamidino-2-phenylindole (not shown).



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 2. Immunofluorescent localization of tight and adherens junction proteins in QL{alpha}12-expressing MDCK cells. Images were obtained before (+dox) and after (-dox) induction of QL{alpha}12 expression for 48 h. Cells were stained for ZO-1 (A and B), claudin-1 (C and D), occludin (E and F), {beta}-catenin (G and H), and E-cadherin (I and J). Scale bar, 25 µm.

 


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. Analysis of Src activation, {beta}-catenin tyrosine phosphorylation, and Rho activation in QL{alpha}12-expressing MDCK cells. A: Western blot of Src in MDCK cells after QL{alpha}12 (QL) or wt{alpha}12 (wt) expression. Whole cell lysates were resolved by SDS-PAGE, and Src was detected by Western blot using anti-c-Src antibodies (left), antiphospho-Tyr419 (pTyr419)-Src antibodies (pY419, middle), or anti-pTyr530-Src antibodies (pY530, right). Uninduced lysates (+dox) were compared with lysates of QL{alpha}12- or wt{alpha}12-expressing MDCK cells (-dox for 48 h). Equal loading of the lanes was shown with occludin antibody after stripping and reblotting of the nitrocellulose. IB, immunoblot. B: tyrosine phosphorylation of immunoprecipitated (IP) {alpha}- and {beta}-catenin. Top: {alpha}- and {beta}-catenin were immunoprecipitated from -dox QL{alpha}12-expressing MDCK cells after 72 h and analyzed by Western blot with {alpha}- or {beta}-catenin antibodies. Bottom: {alpha}-or {beta}-catenin was immunoprecipitated from cell lysates with (-dox) or without (+dox) QL{alpha}12 expression and analyzed by Western blot using anti-pTyr antibody. C: rhotekin-binding assay. Left: Western blot of Rho in cellular lysates and after glutathione S-transferase (GST)-rhotekin pulldown experiments from induced and uninduced wt{alpha}12- and QL{alpha}12-expressing MDCK cells. Uninduced wt{alpha}12- and QL{alpha}12-expressing cell lysates were treated with 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) to irreversibly activate Rho (left) or GDP (100 µM). Glutathione S-transferase-rhotekin pulldown experiments from lysates prepared from uninduced (+dox) or induced (-dox) QL{alpha}12-expressing MDCK cells treated with 100 µM genistein (gen) or 10 µM PP-2 are also shown. Right: Western blot of the same lysates used for pulldown experiments (2% of total lysate). Experiment was performed 3 times with similar results.

 

Epithelial cell polarity is disrupted and cortical actin staining increased with QL{alpha}12 expression. Because expression of QL{alpha}12, but not wt{alpha}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{alpha}12-expressing MDCK cells (Fig. 3A, a and e, and c and g). However, induction of QL{alpha}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{alpha}12 caused no significant changes in the actin cytoskeleton (see Fig. 6A), whereas induction of QL{alpha}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{alpha}12- and wt{alpha}12-expressing MDCK cells in the absence of G{alpha}12 expression (doxycycline-containing medium; see Fig. 6). These findings indicate that QL{alpha}12 expression causes cell rounding, increased stress fibers, and loss of separation between proteins that are normally restricted to specific membrane domains.



View larger version (82K):
[in this window]
[in a new window]
 
Fig. 3. Immunofluorescent localization of ZO-1, Na-K-ATPase, and actin in QL{alpha}12-expressing MDCK cells. A: confocal localization for ZO-1 and Na-K-ATPase in uninduced (+dox) and induced (-dox) QL{alpha}12-expressing MDCK cell monolayers at an apical (a-d) and basal (e-h) plane. B: xz-axis images of uninduced (+dox) and induced (-dox) QL{alpha}12-expressing MDCK cells stained for ZO-1 (a and b), Na-K-ATPase (c and d), and actin (e and f). Note altered distribution of these proteins in QL{alpha}12 (-dox)-expressing cells (b, d, and f), including some apical localization of Na-K-ATPase (arrow, d). Arrows in b and d indicate apical staining. Scale bars, 15 µm.

 


View larger version (103K):
[in this window]
[in a new window]
 
Fig. 6. Confocal immunofluorescent localization of G{alpha}12 (G12), ZO-1, and actin in wt{alpha}12- and QL{alpha}12-expressing cells in the presence of tyrosine kinase inhibitors. A: wt{alpha}12 expression was induced for 72 h (d-l) in the absence (d-f) or presence of 100 µM genistein (g-i) or 10 µM PP-2 (j-l). Cells were stained for G{alpha}12 (a-c), ZO-1 (d-f), and actin (g-i). B: QL{alpha}12 expression was induced for 72 h (d-l) in the absence (d-f) or presence of 100 µM genistein (g-i) or 10 µM PP-2 (j-l). Scale bar, 20 µm.

 

Paracellular regulation of MDCK cell junctions by G{alpha}12 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{alpha}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{alpha}12-expressing MDCK cells (Fig. 4A). Baseline TER in Tet-Off MDCK cells was ~75 {Omega}·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{alpha}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{alpha}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{alpha}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{alpha}12-expressing MDCK cells in the presence and absence of G{alpha}12 expression and inhibitors. Figure 4B demonstrates a large (~7-fold) increase in paracellular flux with induction of QL{alpha}12 expression. The increase in flux from QL{alpha}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{alpha}12-overexpressing MDCK cells. There was a small increase in mannitol flux that was also blocked by the inhibitors (Fig. 4B).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Barrier function in QL{alpha}12-expressing MDCK cells. A: transepithelial resistance (TER) was measured in confluent, uninduced monolayers at the beginning (0 h) and throughout QL{alpha}12 induction (-dox) and resuppression (+dox). An identical protocol was followed in the presence of tyrosine kinase inhibitors genistein (Gen, 100 µM) or PP-2 (10 µM). Tyrosine inhibitors were added at the beginning of QL{alpha}12 induction (0 h) and were present throughout the experiment. At 124 h, tyrosine kinase-treated monolayers were induced again, but without tyrosine kinase inhibitors (-dox). A simultaneous control of uninduced QL{alpha}12-expressing MDCK cells [doxycycline present at all times (+dox only)] is shown for comparison (n = 7 for each time point). B: mannitol flux in wild-type and QL{alpha}12-expressing MDCK cells with and without inhibitors. Cells were cultured on 6-mm Transwell filters for 72 h in the presence or absence of doxycycline and inhibitor. Fluxes of [3H]mannitol were measured at 0, 30, 60, 120, and 180 min, and rates were determined. A representative experiment is shown with n = 3 for each condition, and results are expressed as means ± SD. Experiment was repeated 3-4 times with similar results.

 

Src is activated and {beta}-catenin phosphorylation increased without detectable Rho activation in QL{alpha}12-expressing MDCK cells. On the basis of the observation that PP-2 blocked the QL{alpha}12 protein effects on the MDCK cell junction and the report that Src is activated by G{alpha}12 (28, 41), we investigated c-Src activity in QL{alpha}12- and wt{alpha}12-expressing cells. Western blots of wt{alpha}12- and QL{alpha}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{alpha}12 expression (doxycycline-containing medium), phosphorylation of pTyr419 (as detected with pTyr419-specific antibody) was minimal in wt{alpha}12- and QL{alpha}12-expressing MDCK cells, reflecting Src inactivation (Fig. 5A, middle). Inducing QL{alpha}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{alpha}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{alpha}12 expression, whereas there was no significant difference in pTyr530 Src phosphorylation relative to occludin in wt{alpha}12-expressing cells (Fig. 5A, right).

Our confocal studies showed that E-cadherin and {beta}-catenin localization were disrupted in QL{alpha}12-expressing MDCK cells (Fig. 2). G{alpha}12 recently was demonstrated to interact with E-cadherin and regulate {beta}-catenin release (35), and other studies have shown that {beta}-catenin tyrosine phosphorylation is increased during adherens junction disassembly (72). To determine whether QL{alpha}12 expression caused increased phosphorylation of {beta}-catenin, we immunoprecipitated {alpha}- and {beta}-catenin from QL{alpha}12-expressing MDCK cells in medium with and without doxycycline. In our gel system, {alpha}- and {beta}-catenin migrate with nearly identical molecular mass of 98 kDa. Nevertheless, Fig. 5B (top) shows that {alpha}-catenin precipitates a significant amount of {beta}-catenin, whereas the {beta}-catenin coprecipitates a much smaller amount of {alpha}-catenin. Both {alpha}- and {beta}-catenin were immunoprecipitated from cells in medium with and without doxycycline and probed by Western blot with anti-pTyr antibodies (Fig. 5B, bottom). For {alpha}-and {beta}-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 {beta}-catenin, although we cannot rule out changes in {alpha}-catenin as well. Similar results were obtained when filters were stripped and reprobed with catenin or pTyr antibodies.

Because G{alpha}12 can activate Rho through Src-dependent and Src-independent mechanisms, we determined whether Rho was activated with QL{alpha}12 expression. G{alpha}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{alpha}12-or QL{alpha}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{alpha}12-expressing MDCK cells with the cell-permeant Rho kinase inhibitor Y-27632 had no effect on QL{alpha}12-induced changes in the junction (results not shown).

Tyrosine kinase inhibitors prevent disruption of junctional proteins in QL{alpha}12-expressing MDCK cells. Confocal analysis of QL{alpha}12-expressing MDCK cells reveals that expression of wt{alpha}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{alpha}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{alpha}12 staining pattern. Actin staining was unchanged by wt{alpha}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{alpha}12-expressing MDCK cells, the inhibitors had no major effect on QL{alpha}12 localization, but in QL{alpha}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{alpha}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{alpha}12-expressing MDCK cells (Fig. 6A, h). Unlike wt{alpha}12-expressing MDCK cells, actin staining of QL{alpha}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{alpha}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.



View larger version (82K):
[in this window]
[in a new window]
 
Fig. 7. Actin staining at the level of ZO-1 in QL{alpha}12-expressing MDCK cells. QL{alpha}12-expressing MDCK cells were cultured on Transwell filters for 72 h in doxycycline-containing medium and then switched to doxycycline-free medium in the presence and absence of 100 µM genistein or 10 µM PP-2 for an additional 72 h. Cells were fixed in 4% paraformaldehyde and costained with ZO-1 and phalloidin. Images were obtained at the level of ZO-1 using a Nikon Labophot-2 microscope and Spot Digital camera and software (www.diaginc.com/SpotSoftware/spotrt.htm; version 3.5.7). Images were then processed in Adobe Photoshop. Scale bar, 10 µm.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The regulation of the epithelial cell barrier is appreciated to be a complex and highly regulated process. Multiple signaling mechanisms, including heterotrimeric G proteins, monomeric G proteins, serine-threonine protein kinases, tyrosine kinases, and regulated calcium stores, contribute in interconnecting pathways to maintain an intact junction and regulate its biogenesis (for review see Refs. 2, 9, and 14). Ultimately establishing the direct protein interactions and signaling pathways that regulate these processes will require a combination of protein biochemistry, cell culture models, and transgenic animals. Utilizing in vitro binding techniques and immunoprecipitations from cultured MDCK cells, we recently demonstrated a direct interaction between G{alpha}12 and the tight junction scaffolding protein ZO-1 (37). In addition, we showed that expression of activated G{alpha}12 (QL{alpha}12) in these cells reversibly disrupted the localization of the tight junction protein ZO-1 and increased paracellular permeability as measured by TER and paracellular flux (37). The findings reported here indicate that proteins of the tight and adherens junctions are disrupted, and there is significant reorganization of the actin cytoskeleton. These G{alpha}12-mediated changes appear to be predominantly regulated through Src tyrosine kinases.

Expression of QL{alpha}12 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{alpha}12 on dome formation was reversible, indicating that QL{alpha}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{alpha}12, but not wt{alpha}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 {beta}-catenin. This effect of QL{alpha}12 on junctional proteins appears to be unique within the G protein family. Previous studies expressing wild-type or constitutively active mutants of G{alpha}o, G{alpha}i2, or G{alpha}s had no effect on the staining patterns of ZO-1, occludin, or E-cadherin (10, 49, 50). In the QL{alpha}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{alpha}12, we speculate that the QL{alpha}12-induced changes in ZO-1 localization resulted in the disrupted claudin distribution. However, we cannot exclude the possibility that QL{alpha}12 simultaneously disrupts the adherens junction, because tight junction assembly starts with interactions of E-cadherin and E-cadherin also interacts with G{alpha}12 (39). Similarly, we found the normal separation of ZO-1 and Na-K-ATPase disrupted in QL{alpha}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{alpha}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 G{alpha}i2 and G{alpha}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{alpha}12. The tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 prevented development of the typical phenotype in QL{alpha}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{alpha}12-expressing cells. These studies were not designed to distinguish between direct Src activation by G{alpha}12 and indirect activation of Src via another mechanism. G{alpha}12 directly regulates other tyrosine kinases [Bruton's tyrosine kinase and pp72syk (24, 28)], and several receptors (thrombin, endothelin, and vasopressin) signal through G{alpha}12 to indirectly activate Src (6). Furthermore, direct Src kinase activation by G{alpha}12/13 was shown in thrombin-stimulated platelets and coincided with platelet shape changes (28). Although G{alpha}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{alpha}12 expression. It is also possible that other small G proteins (not detected with rhotekin binding), such as Rac1, are downstream of G{alpha}12 in this system. In an analogous experimental design, Postma et al. (45) showed that G{alpha}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 {beta}-catenin from other mechanisms causing increased phosphorylation (e.g., changes in E-cadherin and catenin localization induced by QL{alpha}12 expression). Future experiments are needed to determine whether the increased {beta}-catenin phosphorylation is important to the G{alpha}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 {beta}-catenin but not {alpha}-catenin (56). This raises the possibility that Src could be directly phosphorylating {beta}-catenin in QL{alpha}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 G{alpha}12 and ZO-1 in vitro and in cells and now show that Src tyrosine kinases are important for the G{alpha}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{alpha}12 have been unsuccessful. This suggests to us that Src is in proximity to ZO-1 and G{alpha}12 but may be in direct association with one of the other scaffolding proteins. Our previous results with activated G{alpha}i2 and G{alpha}s in MDCK cells have shown increased rates of tight junction assembly in the calcium switch model in addition to increased baseline TER. G{alpha}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.


    DISCLOSURES
 
This work was supported by National Institute of General Medical Sciences Grant GM-55223 (to B. M. Denker), National Institute of Diabetes and Digestive and Kidney Diseases National Research Service Award DK-62678 (to J. L. Hunt), a Clinical Scientist Award from the National Kidney Foundation (to B. M. Denker), and Deutsche-Forschungsgemeinschaft (Bonn, Germany) Grant Me 1760/1-1 (to T. N. Meyer).


    ACKNOWLEDGMENTS
 
Present address of T. N. Meyer and C. Schwesinger: Universitötsklinik Hamburg-Eppendorf, Dept. of Internal Medicine, Div. of Nephrology, Martinistrasse 52, 20246 Hamburg, Germany.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. M. Denker, Renal Division, Brigham and Women's Hospital and Harvard Medical School, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. (E-mail: bdenker{at}rics.bwh.harvard.edu).

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


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Balda MS, Gonzalez-Mariscal L, Macias-Silva M, Torres-Marquez ME, Garcia Sainz JA, and Cereijido M. Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol 122: 193-202, 1991.[ISI][Medline]

2. Balda MS and Matter K. Tight junctions. J Cell Sci 111: 541-547, 1998.[Abstract/Free Full Text]

3. Buhl AM, Johnson NL, Dhanasekaran N, and Johnson GL. G{alpha}12 and G{alpha}13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly. J Biol Chem 270: 24631-24634, 1995.[Abstract/Free Full Text]

4. Cereijido M, Ehrenfeld J, Fernandez-Castelo S, and Meza I. Fluxes, junctions, and blisters in cultured monolayers of epithelioid cells (MDCK). Ann NY Acad Sci 372: 422-441, 1981.[Abstract]

5. Chen Y, Lu Q, Schneeberger EE, and Goodenough DA. Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell 11: 849-862, 2000.[Abstract/Free Full Text]

6. Clark EA and Brugge JS. Redistribution of activated pp60csrc to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets. Mol Cell Biol 13: 1863-1871, 1993.[Abstract]

7. Collins LR, Minden A, Karin M, and Brown JH. G{alpha}12 stimulates c-Jun NH2-terminal kinase through the small G proteins Ras and Rac. J Biol Chem 271: 17349-17353, 1996.[Abstract/Free Full Text]

8. De Almeida JB, Holtzman EJ, Peters P, Ercolani L, Ausiello DA, and Stow JL. Targeting of chimeric G{alpha}i proteins to specific membrane domains. J Cell Sci 107: 507-515, 1994.[Abstract/Free Full Text]

9. Denker BM and Nigam SK. Molecular structure and assembly of the tight junction. Am J Physiol Renal Physiol 274: F1-F9, 1998.[Abstract/Free Full Text]

10. Denker BM, Saha C, Khawaja S, and Nigam SK. Involvement of a heterotrimeric G protein {alpha}-subunit in tight junction biogenesis. J Biol Chem 271: 25750-25753, 1996.[Abstract/Free Full Text]

11. Dodane V and Kachar B. Identification of isoforms of G proteins and PKC that colocalize with tight junctions. J Membr Biol 149: 199-209, 1996.[ISI][Medline]

12. Fanning AS and Anderson JM. PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane. J Clin Invest 103: 767-772, 1999.[Free Full Text]

13. Fanning AS and Anderson JM. Protein modules as organizers of membrane structure. Curr Opin Cell Biol 11: 432-439, 1999.[ISI][Medline]

14. Fanning AS, Mitic LL, and Anderson JM. Transmembrane proteins in the tight junction barrier. J Am Soc Nephrol 10: 1337-1345, 1999.[Abstract/Free Full Text]

15. Fromm C, Coso OA, Montaner S, Xu N, and Gutkind JS. The small GTP-binding protein Rho links G protein-coupled receptors and G{alpha}12 to the serum response element and to cellular transformation. Proc Natl Acad Sci USA 94: 10098-10103, 1997.[Abstract/Free Full Text]

16. Fukuhara S, Murga C, Zohar M, Igishi T, and Gutkind JS. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J Biol Chem 274: 5868-5879, 1999.[Abstract/Free Full Text]

17. Gohla A, Harhammer R, and Schultz G. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J Biol Chem 273: 4653-4659, 1998.[Abstract/Free Full Text]

18. Gohla A, Offermanns S, Wilkie TM, and Schultz G. Differential involvement of G{alpha}12 and G{alpha}13 in receptor-mediated stress fiber formation. J Biol Chem 274: 17901-17907, 1999.[Abstract/Free Full Text]

19. Gomez S, del Mont Llosas M, Verdu J, Roura S, Lloreta J, Fabre M, and Garcia de Herreros A. Independent regulation of adherens and tight junctions by tyrosine phosphorylation in Caco-2 cells. Biochim Biophys Acta 1452: 121-132, 1999.[ISI][Medline]

20. Gonzalez-Mariscal L, Contreras RG, Bolivar JJ, Ponce A, Chavez De Ramirez B, and Cereijido M. Role of calcium in tight junction formation between epithelial cells. Am J Physiol Cell Physiol 259: C978-C986, 1990.[Abstract/Free Full Text]

21. Hamilton SE and Nathanson NM. Differential localization of G proteins, G{alpha}o and G{alpha}i1, -2, and -3 in polarized epithelial MDCK cells. Biochem Biophys Res Commun 234: 1-7, 1997.[ISI][Medline]

22. Itoh M, Furuse M, Morita K, Kubota K, Saitou M, and Tsukita S. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol 147: 1351-1363, 1999.[Abstract/Free Full Text]

23. Jiang H, Wu D, and Simon MI. The transforming activity of activated G{alpha}12. FEBS Lett 330: 319-322, 1993.[ISI][Medline]

24. Jiang Y, Ma W, Wan Y, Kozasa T, Hattori S, and Huang XY. The G protein G{alpha}12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain. Nature 395: 808-813, 1998.[ISI][Medline]

25. Jou TS and Nelson WJ. Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity. J Cell Biol 142: 85-100, 1998.[Abstract/Free Full Text]

26. Jou TS, Schneeberger EE, and Nelson WJ. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol 142: 101-115, 1998.[Abstract/Free Full Text]

27. Katoh H, Aoki J, Yamaguchi Y, Kitano Y, Ichikawa A, and Negishi M. Constitutively active G{alpha}12, G{alpha}13, and G{alpha}q induce Rho-dependent neurite retraction through different signaling pathways. J Biol Chem 273: 28700-28707, 1998.[Abstract/Free Full Text]

28. Klages B, Brandt U, Simon MI, Schultz G, and Offermanns S. Activation of G12/G13 results in shape change and Rho/Rhokinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 144: 745-754, 1999.[Abstract/Free Full Text]

29. Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, and Sternweis PC. p115 RhoGEF, a GTPase activating protein for G{alpha}12 and G{alpha}13. Science 280: 2109-2111, 1998.[Abstract/Free Full Text]

30. Leighton J, Brada Z, Estes LW, and Justh G. Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science 163: 472-473, 1969.[ISI][Medline]

31. Majumdar M, Seasholtz TM, Buckmaster C, Toksoz D, and Brown JH. A Rho exchange factor mediates thrombin and G{alpha}12-induced cytoskeletal responses. J Biol Chem 274: 26815-26821, 1999.[Abstract/Free Full Text]

32. Mao J, Xie W, Yuan H, Simon MI, Mano H, and Wu D. Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by G{alpha}12/13. EMBO J 17: 5638-5646, 1998.[Abstract/Free Full Text]

33. Martinez-Estrada OM, Villa A, Breviario F, Orsenigo F, Dejana E, and Bazzoni G. Association of junctional adhesion molecule with calcium/calmodulin-dependent serine protein kinase (CASK/LIN-2) in human epithelial Caco-2 cells. J Biol Chem 276: 9291-9296, 2001.[Abstract/Free Full Text]

34. McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, and Schneeberger EE. Occludin is a functional component of the tight junction. J Cell Sci 109: 2287-2298, 1996.[Abstract/Free Full Text]

35. Meigs TE, Fedor-Chaiken M, Kaplan DD, Brackenbury R, and Casey PJ. G{alpha}12 and G{alpha}13 negatively regulate the adhesive functions of cadherin. J Biol Chem 277: 24594-24600, 2002.[Abstract/Free Full Text]

36. Meigs TE, Fields TA, McKee DD, and Casey PJ. Interaction of G{alpha}12 and G{alpha}13 with the cytoplasmic domain of cadherin provides a mechanism for {beta}-catenin release. Proc Natl Acad Sci USA 98: 519-524, 2001.[Abstract/Free Full Text]

37. Meyer TN, Schwesinger C, and Denker BM. Zonula occludens-1 is a scaffolding protein for signaling molecules. G{alpha}12 directly binds to the Src homology 3 domain and regulates paracellular permeability in epithelial cells. J Biol Chem 277: 24855-24858, 2002.[Abstract/Free Full Text]

38. Meyer TN, Schwesinger C, Ye J, Denker BM, and Nigam SK. Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. J Biol Chem 276: 22048-22055, 2001.[Abstract/Free Full Text]

39. Mitic LL and Anderson JM. Molecular architecture of tight junctions. Annu Rev Physiol 60: 121-142, 1998.[ISI][Medline]

40. Mullin JM, Kampherstein JA, Laughlin KV, Clarkin CE, Miller RD, Szallasi Z, Kachar B, Soler AP, and Rosson D. Overexpression of protein kinase C{delta} increases tight junction permeability in LLC-PK1 epithelia. Am J Physiol Cell Physiol 275: C544-C554, 1998.[Abstract/Free Full Text]

41. Nagao M, Kaziro Y, and Itoh H. The Src family tyrosine kinase is involved in Rho-dependent activation of c-Jun N-terminal kinase by G{alpha}12. Oncogene 18: 4425-4434, 1999.[ISI][Medline]

42. Neer EJ. Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80: 249-257, 1995.[ISI][Medline]

43. Patrie KM, Drescher AJ, Welihinda A, Mundel P, and Margolis B. Interaction of two actin-binding proteins, synaptopodin and {alpha}-actinin-4, with the tight junction protein MAGI-1. J Biol Chem 277: 30183-30190, 2002.[Abstract/Free Full Text]

44. Plonk SG, Park SK, and Exton JH. The {alpha}-subunit of the heterotrimeric G protein G13 activates a phospholipase D isozyme by a pathway requiring Rho family GTPases. J Biol Chem 273: 4823-4826, 1998.[Abstract/Free Full Text]

45. Postma FR, Hengeveld T, Alblas J, Giepmans BN, Zondag GC, Jalink K, and Moolenaar WH. Acute loss of cell-cell communication caused by G protein-coupled receptors: a critical role for c-Src. J Cell Biol 140: 1199-1209, 1998.[Abstract/Free Full Text]

46. Prasad MV, Dermott JM, Heasley LE, Johnson GL, and Dhanasekaran N. Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of G{alpha}12 and G{alpha}13. J Biol Chem 270: 18655-18659, 1995.[Abstract/Free Full Text]

47. Ren XD, Kiosses WB, and Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18: 578-585, 1999.[Abstract/Free Full Text]

48. Rosson D, O'Brien TG, Kampherstein JA, Szallasi Z, Bogi K, Blumberg PM, and Mullin JM. Protein kinase C-{alpha} activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line. J Biol Chem 272: 14950-14953, 1997.[Abstract/Free Full Text]

49. Saha C, Nigam SK, and Denker BM. Expanding role of G proteins in tight junction regulation: G{alpha}s stimulates TJ assembly. Biochem Biophys Res Commun 285: 250-256, 2001.[ISI][Medline]

50. Saha C, Nigam SK, and Denker BM. Involvement of G{alpha}i2 in the maintenance and biogenesis of epithelial cell tight junctions. J Biol Chem 273: 21629-21633, 1998.[Abstract/Free Full Text]

51. Schindler T, Sicheri F, Pico A, Gazit A, Levitzki A, and Kuriyan J. Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. Mol Cell 3: 639-648, 1999.[ISI][Medline]

52. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, and Lifton RP. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 285: 103-106, 1999.[Abstract/Free Full Text]

53. Slice LW, Bui L, Mak C, and Walsh JH. Differential regulation of COX-2 transcription by Ras- and Rho-family of GTPases. Biochem Biophys Res Commun 276: 406-410, 2000.[ISI][Medline]

54. Strathmann MP and Simon MI. G{alpha}12 and G{alpha}13 subunits define a fourth class of G protein {alpha}-subunits. Proc Natl Acad Sci USA 88: 5582-5586, 1991.[Abstract]

55. Stuart RO, Sun A, Panichas M, Hebert SC, Brenner BM, and Nigam SK. Critical role for intracellular calcium in tight junction biogenesis. J Cell Physiol 159: 423-433, 1994.[ISI][Medline]

56. Takeda H and Tsukita S. Effects of tyrosine phosphorylation on tight junctions in temperature-sensitive v-src-transfected MDCK cells. Cell Struct Funct 20: 387-393, 1995.[ISI][Medline]

57. Tanaka A and Fujita DJ. Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector. Mol Cell Biol 6: 3900-3909, 1986.[ISI][Medline]

58. Thomas SM and Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13: 513-609, 1997.[ISI][Medline]

59. Tsukamoto T and Nigam SK. Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins. Am J Physiol Renal Physiol 276: F737-F750, 1999.[Abstract/Free Full Text]

60. Tsukita S, Furuse M, and Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2: 285-293, 2001.[ISI][Medline]

61. Tsukita S, Oishi K, Akiyama T, Yamanashi Y, Yamamoto T, and Tsukita S. Specific proto-oncogenic tyrosine kinase of Src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J Cell Biol 113: 867-879, 1991.[Abstract]

62. Van Itallie C, Rahner C, and Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 107: 1319-1327, 2001.[Abstract/Free Full Text]

63. Voyno-Yasenetskaya TA, Pace AM, and Bourne HR. Mutant {alpha}-subunits of G12 and G13 proteins induce neoplastic transformation of Rat-1 fibroblasts. Oncogene 9: 2559-2565, 1994.[ISI][Medline]

64. Wadsworth SJ, Gebauer G, van Rossum GD, and Dhanasekaran N. Ras-dependent signaling by the GTPase-deficient mutant of G{alpha}12. J Biol Chem 272: 28829-28832, 1997.[Abstract/Free Full Text]

65. Walsh SV, Hopkins AM, Chen J, Narumiya S, Parkos CA, and Nusrat A. Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia. Gastroenterology 121: 566-579, 2001.[ISI][Medline]

66. Xu N, Bradley L, Ambdukar I, and Gutkind JS. A mutant {alpha}-subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells. Proc Natl Acad Sci USA 90: 6741-6745, 1993.[Abstract]