April 2002
Volume 43, Issue 4
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Cornea  |   April 2002
Rho and Rho-Kinase (ROCK) Signaling in Adherens and Gap Junction Assembly in Corneal Epithelium
Author Affiliations
  • Susan C. Anderson
    From the Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
  • Cynthia Stone
    From the Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
  • Lisa Tkach
    From the Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
  • Nirmala SundarRaj
    From the Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Investigative Ophthalmology & Visual Science April 2002, Vol.43, 978-986. doi:https://doi.org/
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      Susan C. Anderson, Cynthia Stone, Lisa Tkach, Nirmala SundarRaj; Rho and Rho-Kinase (ROCK) Signaling in Adherens and Gap Junction Assembly in Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2002;43(4):978-986. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To examine whether Rho and its downstream target, a Rho-associated kinase (ROCK), are involved in the regulation of the assembly of cadherin-mediated cell adhesion and connexin 43 (Cx43) gap junctions in corneal epithelium.

methods. Rho and ROCK activities in rabbit corneal epithelial cells in culture were inhibited by microinjection of a Clostridium botulinum ADP-ribosyltransferase (C3) and treatment with a ROCK specific inhibitor (Y-27632), respectively. Immunocytochemical and Western blot techniques were used to study the distribution and relative concentrations of E-cadherin and Cx43. Intercellular communication via gap junctions was measured by a dye transfer assay.

results. Inhibition of Rho activity in the primary cultures of rabbit corneal epithelial cells by microinjecting them with C3 resulted in an inhibition of the assembly of E-cadherin-based cell-cell adhesion and Cx43 gap junctions. However, inhibition of the ROCK activity by treatment with Y-27632 inhibited the assembly of E-cadherin-based cell-cell adhesions but not Cx43 gap junctions. In fact, inhibition of ROCK resulted in an increase in the number of Cx43 gap junctions and in cell-cell communication. Culturing corneal epithelial cells in a low calcium medium prevented the formation of E-cadherin adherens junctions but not the Cx43 gap junctions.

conclusions. E-cadherin adherens junctions are not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Different Rho signaling pathways are involved in the regulation of the assembly of E-cadherin mediated cell-cell adhesion and Cx43 gap junctions. Although a Rho/ROCK signaling pathway influences the assembly of E-cadherin adherens junctions, its downregulation influences Cx43 gap junction assembly.

Cells, in most tissues, communicate through gap junctions that are intercellular hydrophilic channels that allow the transfer of cytoplasmic molecules of <1 kDa between neighboring cells. 1 2 3 Gap junctions have been implicated to be important in the control of cell proliferation, differentiation, and regeneration. 4 5 They are composed of connexins (Cxs), a family of related transmembrane proteins. 2 After their synthesis in the endoplasmic reticulum, Cxs oligomerize into hexameric hemichannels (connexons) in the Golgi and are transported to the cell surface. Gap junction channels, connecting the cytoplasm of the adjoining cells, are formed by an interaction of the transmembrane connexon from one cell with the connexon of the adjoining cell. The turnover time of Cx has been reported to be very short, with a half-life of only 1.3–2 hours. 6 7 8 The mechanisms of the transport of connexins to the cell membranes from the Golgi and the alignment of opposing connexons to form the gap junction channels are poorly understood, and the possible interacting molecules involved in the assembly of gap junctions have not yet been identified. Several reported findings suggest that the formation of cadherin-based adherens junctions precedes and facilitates the assembly of connexons at the cell-cell contact regions. 9 10 11 12 13 14 For example, the inhibition of E-cadherin- or N-cadherin-based cell-cell adhesion, using antibodies to these proteins, has been shown to inhibit the assembly of gap junctions. 9 In poorly coupled cells, the expression of recombinant cadherins was shown to greatly increase coupling. 11 The expression of cadherin did not affect the synthesis of Cxs but increased the phosphorylation of Cx and Cx gap junctions at the cell-cell contacts. 12 Immunoelectron microscopic analyses showed a colocalization of connexin, E-cadherin, and β-catenin at the cell-cell contact sites during gap junction formation. 15 A more recent report suggests that the effect of cadherins on gap junction assembly is cell-type specific. 16 Although an increase in cadherin-based adhesion resulted in an increase in the gap junction communication in hepatoma cells in culture, it has an opposite effect in the L cells. E-cadherin has been shown to be involved in controlling the specificity of gap junction formation. When rat epithelial cells expressing P- and 125-kDa N-cadherin are grown in a mixed culture with rat fibroblasts expressing 140-kDa N-cadherin, each cell type established homologous communication via Cx43 gap junctions and very little heterologous communication. However, transfection of both these cells with E-cadherin resulted in a 10-fold increase in heterologous communication. 17  
Cadherin-mediated cell-cell adhesion is formed by the homophilic interaction of the extracellular domains of the cadherins of the adjacent cells and the interaction of the cytoplasmic domain with catenins (β-catenin, γ-catenin/plakoglobin, and α-catenin) that link E-cadherin to the actin cytoskeleton. 18 19 20 The Rho family of small GTPases, including Rho, Rac, and Cdc42, have been implicated in the development and maintenance of E-cadherin-mediated cell-cell adhesions. 21 22 23 24 25 These GTPases are now well known for their regulation of distinct patterns of actin filament organization and a wide range of actin-based cellular processes. 26 27 28 29 In MDCK cells, inhibition of RhoA, Rac, or Cdc42 activity has been found to result in the loss of E-cadherin-mediated cell-cell adhesion. 30 31 32 In keratinocytes, the inhibition of Rac or RhoA activity has been shown to inhibit the induction of Ca2+-dependent E-cadherin-mediated cell-cell adhesion. 25 33 Based on the current information, it is not clear whether the involvement of specific Rho family members in the adherens interactions, mediated by specific cadherins, is cell-type specific. The knowledge of the downstream events leading to adherens junction formation, which may be a prerequisite for gap junction formation, is also limited. Whether the requirement of adherens interactions in gap junction formation is tissue-type dependent has not been investigated. If the gap junction formation were dependent on cadherin-based adherens junction formation, which in turn was regulated by one or more members of the Rho family, then these GTPases should also regulate the gap junction assembly. Although the corneal epithelium has Cx43 gap junctions, 34 35 the surrounding limbal epithelium, which harbors the stem cells of corneal epithelium, lacks Cx43 gap junctions. 36 Coincidently, an isoform of Rho-associated kinase, ROCK-I, is also absent in the limbal epithelium. The study reported here evaluated whether the Rho signaling cascades, specifically the signaling pathway(s) involving ROCK, 37 38 39 40 were involved in the formation of E-cadherin adherens junctions and whether E-cadherin adherens junction formation was a prerequisite for Cx43 gap junction assembly in corneal epithelial cells. 
Materials and Methods
Cell Culture and Treatments
All procedures involving rabbits were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Corneas with the adjacent limbus were excised from rabbit eyes (Pel-Freez Biologicals, Rogers, AK) and used for growing the primary cultures (P0), in SHEM (supplemental hormonal epithelial medium 41 ) according to Ebato et al. 42 Cells in P0 were subcultured, using 0.25% trypsin/EDTA (GIBCO-BRL, Grand Island, NY), into 60-mm tissue culture dishes or four-well chamber tissue culture slides (Nalge-Nunc International, Napersville, IL) at a density of 3 × 104 cells/cm2 and incubated further to allow them to reach a desired density. 
When identical sets of P1 cells grown in either 35- or 60-mm tissue culture dishes or in chamber slides had reached confluency, they were treated with the 10 μM ROCK inhibitor, Y-27632 (Welfide Pharmaceutical Industries, Osaka, Japan), in SHEM medium or with SHEM without the inhibitor (control). After a further incubation of cells in these media for specific lengths of time, the cells were processed for either immunostaining or for Western blot analyses. Media with or without inhibitor were changed every 6 hours during the treatments. 
To grow the cells in low calcium (Ca2+) medium, P0 cells growing in SHEM with normal Ca2+ concentration (1.8 mM) were subcultured into low Ca2+ (5 μM) medium (DMEM/HAM F12-deficient medium with 2.5 mM glutamine, 0.45 mM leucine, 0.5 mM lysine, 0.14 mM MgCl2, 0.2 mM MgSO4 and 5 μM Ca2+ chloride), 10% dialyzed fetal bovine serum and penicillin/streptomycin. After the cells had reached confluency, one set of cultures was processed for immunofluorescence and Western blot analyses and in the remaining dishes, culture medium was replaced with SHEM with or without 10 μM Y-27632 43 and normal levels of Ca2+ (controls). After an additional 4 hours of incubation, the cells were processed as above. 
Microinjections
Microinjections were carried out using freshly pulled needles, a Leitz micromanipulator (Deerfield, IL), and a Narashige microinjector (East Meadow, NY) set at a continuous positive injection pressure of 5 to 7 psi. C3 (Clostridium botulinum ADP-ribosyltransferase; Cytoskeleton Inc., Denver, CO) at a concentration of 0.1 mg/mL with 0.4 mg/mL of rhodamine-dextran, M r 10 × 103 (Molecular Probes, Eugene, OR), in a buffer containing 20 mM Tris-HCl, 20 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7.4, was injected into the cytoplasm of the corneal epithelial cells in primary explant cultures, grown in 60-mm tissue culture dishes. After 4 or 6 hours of incubation, the cells were fixed and immunoreacted with anti-Cx43 or anti-E-cadherin antibodies as described below. 
Immunostaining
Cultures grown in chamber slides or tissue culture dishes and treated as above were rinsed three times with PBS, fixed with 2% paraformaldehyde-lysine-periodate fixative for 5 minutes, and permeabilized with a buffer containing 0.2% Triton X-100 according to McLean and Nakane. 44 The fixed cells were reacted with either 1.5% (for Cx43 staining) or 10% (for E-cadherin staining) heat-inactivated goat serum in phosphate-buffered saline, pH 7.5 (PBS), for 45 minutes to block the nonspecific binding of the secondary antibody, rinsed with PBS, and then treated with the primary and secondary antibodies using a previously described technique. 45 The primary antibodies included rabbit anti-Cx43 (Zymed Laboratory, Inc., San Francisco, CA) at 1:100 concentration and mouse monoclonal anti-E-cadherin (BD Transduction Laboratories, Lexington, KY) at 5 μg/mL concentration, and the secondary antibodies were Alexa 488-conjugated goat anti-rabbit or anti-mouse IgG (Molecular Probes), at 1:1500 or 1:2500 concentration, respectively. For nuclear staining, the immunostained cells were treated with 5 μg/mL of propidium iodide in PBS for 30 seconds, rinsed with PBS, and mounted in Immuno-mount (Shandon, Pittsburgh, PA). In the double fluorescence analyses, the red and green fluorescent Z-stack images (0.25-μm interval) were collected sequentially using a Bio-Rad Radiance 2000 (Hertfordshire, UK) confocal scanning laser system attached to an Olympus IX70 inverted microscope (Tokyo, Japan). 
To visualize the organization of the actin filaments, the cells were fixed and permeabilized as above and incubated with 1:500 dilution of Texas Red-X-conjugated phalloidin (Molecular Probes) for 45 minutes, washed with PBS, and mounted as above. 
Western Blot Analyses
Cultures grown in 60-mm dishes and treated as described earlier were extracted in a buffer containing 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, pH 7.4, 1% Triton X-100, 0.03 TIU/mL aprotinin (Sigma Chemical Co., St. Louis, MO), 1 mM sodium orthovanadate, and 100 μg/mL phenylmethylsulfonyl fluoride (PMSF), and the insoluble fraction was used for the analysis of Cx43 in the gap junctions. For the analyses of E-cadherin, the cells were extracted in a modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 0.03 TIU/mL aprotinin [Sigma], 1 mM sodium orthovanadate, 2 mM PMSF, 1 μg mL leupeptin, 1 μg/mL pepstatin A, and 5 mM EDTA). Briefly, the cells in the dishes were rinsed with cold PBS and then 0.2 mL of RIPA buffer was added per dish to lyse the cells. The cell lysate was scraped and collected, and the dishes were rinsed with an additional 0.1 mL RIPA buffer, which was mixed with the first lysate. The lysate was then passed through a 21-gauge needle and then centrifuged at 10,000g for 20 minutes at 4°C. The proteins in the supernatants were estimated using BCA protein assay reagent (Pierce, Rockford, IL). Aliquots of samples containing 20 μg of proteins were subjected to 12% or 7% SDS-PAGE for Cx43 and E-cadherin, respectively. The proteins separated on the SDS-PAGE were electrophoretically transferred to a nitrocellulose membrane (Schleicher and Schull, Keene, NH), and after treating the membranes with BLOTTO 46 to block the nonspecific binding sites, the blots were immunoreacted with anti-Cx43 (1:250) and anti-E-cadherin (1:250) antibodies and the horseradish peroxidase-conjugated secondary antibodies as described previously. 45 The immunoreactive bands were detected using chemiluminescence reagents (Super Signal West Femto reagent from Pierce), following the manufacturer’s protocols. The chemiluminescent bands detected on the x-ray film were scanned, and the relative differences in their intensities were estimated using the Image-Pro Plus analyses software (Media Cybernetics, Silver Spring, MD) and were normalized with intensities of three major protein bands in duplicate blots stained with Coomassie blue. Data were collected from a minimum of three different sets of experiments and were represented as mean ± SD. The t-test was used to determine whether the differences in the relative intensities of the reactive bands were statistically significant. 
Dye Diffusion
Gap junction permeability was determined using a Lucifer yellow scrapeloading technique. 47 Identical sets of confluent corneal epithelial cells in 35-mm dishes (either treated with Y-27632 for 18 hours or not treated) were rinsed with PBS and then covered with 1.5 mL of 0.05% Lucifer yellow and 1 mg/mL rhodamine dextran, M r 10 × 103 (Molecular Probes) in PBS, and a scrape line was made in the monolayer using a surgical blade. After 5 minutes, the cells were washed with PBS and fixed with 3.7% paraformaldehyde. The fluorescent images were collected using a Bio-Rad Radiance 2000 confocal scanning laser system attached to an Olympus IX70 inverted microscope and the distance of dye diffusion was measured in three sets of experiments using a Image-Pro Plus Image Analysis System (Media Cybernetics, Silver Spring, MD). 
Results
Effects of Rho Inhibition on E-cadherin and Cx43 Distribution in Corneal Epithelial Cells
Immunofluorescence staining of E-cadherin in the primary cultures of rabbit corneal epithelial cells showed that E-cadherin was localized at the lateral plasma membranes. Less intense intracellular staining was also evident (Fig. 1C) . Similarly, Cx43 gap junctions were evident at the cell-cell contacts (Fig. 1G) . To analyze whether Rho GTPase was involved in the formation of E-cadherin adherent junctions and Cx43 gap junctions in the corneal epithelial cells, groups of corneal epithelial cells in the primary cultures were injected with C3 and rhodamine dextran. Immunofluorescence analyses indicated that E-cadherin staining was lost from the lateral membranes of the C3-injected cells within 4 hours (Fig. 1B 1D) but not in the controls injected with rhodamine dextran alone (Fig. 1A 1C) . After 6 hours of incubation, Cx43 staining was significantly reduced in the C3-injected cells (Fig. 1F 1H) but not in the controls (Fig. 1E 1G)
Effects of Inhibition of ROCKs on E-cadherin and Cx43 in Corneal Epithelial Cells
To analyze whether the Rho signaling pathway(s), involving ROCKs (ROCK-I and/or ROCK-II), participates in the formation of E-cadherin adherens junctions and Cx43 gap junctions in corneal epithelial cells, the effects of the inhibition of ROCK with a specific inhibitor, Y-27632, was analyzed in P1 cultures of corneal epithelium. To ensure that ROCK was inactivated in the cells treated with Y-27632, a set of cells was analyzed for the changes in the actin filaments. Within 6 hours of treatment, actin filaments were disrupted in the Y-27632-treated cells, as evident from the staining of the cells with Texas Red-X-conjugated phalloidin (not shown). These cells also developed many long cytoplasmic extensions. These changes remained evident after 12 and 18 hours of inhibitor treatment. E-cadherin at the cell-cell contacts (Fig. 2A) was lost within 6 hours of treatment with the ROCK inhibitor Y-27632 (Fig. 2D) , as evident from the absence of immunostaining, and remained undetectable at the cell-cell contacts after 12 and 18 hours of the inhibitor treatment (Fig. 2E and 2F , respectively). However, cells treated with Y-27632 exhibited cytoplasmic immunostaining of E-cadherin. E-cadherin was probably internalized upon inactivation of ROCK in these cells. Cx43 staining was not lost at the cell-cell contacts after 6 hours of the inhibitor treatment (Fig. 3D) , and after 12 and 18 hours there appeared to be an increase in the Cx43 gap junctions at the lateral surface of the cells (Fig. 3E and 3F , respectively). In addition, significant perinuclear staining, possibly in the Golgi region, and punctate gap junction-like intracellular staining was detected in many of the cells treated with Y-27632 (Fig. 3D 3E 3F)
Western Blot Analyses of the Changes in E-cadherin and Cx43 in Corneal Epithelial Cells
A comparative Western blot analysis of the cell extracts of the Y-27632-treated and nontreated cells was performed to determine the relative levels of E-cadherin and Cx43. Cx43, assembled into gap junctions, has been reported to be insoluble in Triton X-100. 6 The Western blot analyses of the Triton X-100-soluble and -insoluble extracts indicated that most of the Cx43 in the cells, treated or not treated with the inhibitor for 18 hours, was insoluble in Triton X-100 (Fig. 4) . The concentrations of Cx43 in the inhibitor-treated cells were also 1.6 ± 0.252 higher than the nontreated cells. Although the levels of E-cadherin in the control and the cells treated with Y-27632 for 6, 12, and 18 hours were not significantly different (Fig. 5 , top left), the concentration of Cx43 increased upon inactivation of ROCK with Y-27632 (Fig. 5 , top right). From 6 to 18 hours, as the cell density increased, the concentration of Cx43 also increased to varying extents (8–10-fold) in different experiments. However, the concentrations of Cx43 were always higher in the Y-27632-treated cells compared with corresponding concentrations in the nontreated controls. The bar graphs in Figure 5 (bottom) show the relative differences in the intensities of immunoreactive bands in the Western blot analysis of one representative experiment. The intensities of immunoreactive bands in the inhibitor-treated cells, as determined from three different experiments, were 1.5 ± 0.2-, 1.53 ± 0.1-, and 1.6 ± 0.14-fold higher than the controls at 6, 12, and 18 hours, respectively (P < 0.001, P < 0.001, and P < 0.001, respectively). 
Effects of ROCK Inhibition on Intercellular Communication
Inhibition of ROCK activity in the corneal epithelial cells resulted in an increase of the concentration of Cx43 and the number of gap junctions, based on the immunocytochemical and Western blot analyses. To study whether there was a change in intercellular communication, the extent of cell-cell diffusion of Lucifer yellow in the cells treated with Y-27632 for 18 hours was compared with control nontreated cells. Figure 6 shows typical results. There was a 1.36 ± 0.08-fold increase in the distance of the dye diffusion in the cells treated with the inhibitor. In confluent monolayers of corneal epithelial cells, the depth of lateral dye diffusion was 9 or 10 cells in the controls and 13 or 14 cells in the ROCK-inhibited cells. 
Effects of Inhibition of E-cadherin Junctions on Cx43 Gap Junction Formation
The formation of E-cadherin adherens junctions is inhibited in keratinocytes 21 by reducing the concentration of Ca2+ in the culture medium. A similar effect was also evident in P1 corneal epithelial cells grown in low Ca2+ medium (Fig. 7A) . However, Cx43 gap junctions were present in low Ca2+ medium (Fig. 7D) . In another set of cultures, the low Ca2+ medium was replaced with medium containing the normal Ca2+ concentration in the presence or absence (control) of inhibitor, Y-27632. After 4 hours of further incubation, although E-cadherin junctions were assembled and were evident at the lateral surfaces in the controls (Fig. 7B) , they were absent in the cultures treated with Y-27632 (Fig. 7C) . Cx43 junctions were evident in both controls and inhibitor-treated cells (Fig. 7E and 7F , respectively). However, the number of Cx43 gap junctions appeared to be higher in the Y-27632-treated cells. This was confirmed by Western blot analyses (not shown). 
Discussion
Currently, the mechanism of the regulation of gap junction assembly is not well understood. Reportedly, cadherin-mediated cell-cell adhesion is thought to be a prerequisite for the assembly of gap junctions. 9 10 11 12 13 14 Recently, it has become evident that assembly of cadherin-based adherens junctions is regulated by the Rho super family of small GTPases, including Rac, Rho, and Cdc42. 22 23 24 25 Therefore, if Cx43 gap junction assembly were dependent on cadherin junctions, Rho would be involved in the regulation of the assembly of Cx43 gap junctions, and the inhibition of the formation of E-cadherin adherens junctions would result in an inhibition of Cx43 gap junction assembly. This hypothesis was tested in the corneal epithelial cells in the present study. Corneal epithelium is a stratified epithelium like the epidermal epithelium. Epidermal cells 48 as well as corneal epithelial cells 49 express E-cadherin. Gap junctions composed of Cx43 and Cx50 have been identified in corneal epithelium. 34 35 36  
In the present study, the possible involvement of Rho signaling pathway(s), in the formation of E-cadherin-based adherens junctions and the assembly of Cx43 gap junctions were evaluated by studying the consequence of Rho inhibition in corneal epithelial cells. When Rho activity was inhibited in the primary cultures of rabbit corneal epithelial cells by injecting C3, E-cadherin adherens junctions were diminished at cell-cell contact regions, indicating that Rho signaling pathway(s) was involved in their assembly. Rho involvement in the assembly of E-cadherin-based adherens junctions in corneal epithelium conformed with the previously reported findings in the MDCK cell line 30 31 32 and keratinocytes. 50 It was interesting to note that Cx43 gap junctions were also lost in the cells injected with C3, indicating that a Rho-signaling pathway was also involved in Cx43 gap junction assembly in the corneal epithelial cells. Lampe et al. 51 have observed that during the healing of keratinocyte wounds in vitro, interaction of keratinocyte cells with laminin via α3β1 integrin promotes gap junction assembly, and this process requires Rho signaling. 
Rho signaling is involved in the formation of stress fibers and focal adhesions 27 28 29 and consequentially, several actin filament-mediated processes. Recently, several downstream targets of Rho have been identified and among them is a family of isozymes of Rho-associated serine/threonine kinases (collectively referred to as ROCK), including ROCK-I (p160ROCK/ROK-beta) and ROCK-II (ROK-alpha/Rho-kinase). ROCK has been shown to induce focal adhesions and stress fibers in cultured fibroblasts and epithelial cells. 38 40 52 53 Uehata et al. 54 have reported that a new pyridine derivative, Y-27632, is a specific inhibitor of the ROCK family. When ROCK was inhibited by treating corneal epithelial cells with Y-27632, it disrupted actin stress fibers in corneal epithelial cells, as expected. Based on the immunofluorescence analysis, ROCK inhibition resulted in the elimination of E-cadherin-based adherens junctions at the cell-cell contacts. However, it did not inhibit the assembly of Cx43 gap junctions. In fact, inhibition of ROCK activity resulted in an increase in the number of gap junctions, as evident from immunofluorescence and Western blot analyses. There was also an increase in functional gap junctions as evident from the dye diffusion study. We currently do not know whether other gap junctions, such as connexin 50 gap junctions, also accounted for an increase in functional gap junctions in ROCK-inhibited cells. Although E-cadherin was diminished from the cell-cell contacts in Y-27632-treated cells, it was detectable in the cytoplasm of the cells. The total levels of E-cadherin in the inhibitor-treated cells were not significantly different from the controls. Thus, ROCK-I signaling most likely participates in the actin filament-mediated translocation of E-cadherin to the cell membrane. Contrary to the present observation, the inhibition of ROCK with Y-27632 did not prevent clustering of cadherin during induction of cell-cell adhesion in keratinocytes in culture. 33 However, blocking of ROCK function by using a dominant negative approach was reported to partially perturb the localization of cadherin receptors in MDCK cells. 55 In the absence of E-cadherin adherens junctions, Cx43 gap junctions were detected even after 18 hours of inhibitor treatment. E-cadherin is a transmembrane protein, and its adhesive function is brought about by Ca2+-dependent homophilic interaction between E-cadherin molecules of the adjacent cells. The cytoplasmic tail of E-cadherin interacts with the actin cytoskeleton via β-catenin, and this complex formation aides in the clustering of the receptors and strengthening of cell-cell adhesion. 18 19 20 The interaction of the cytoskeleton not only is involved in the clustering of the E-cadherin receptors but also in providing a framework for the different cytoskeletal proteins and signaling molecules at cell-cell junctions. 56 The lowering of the Ca2+ concentration in tissue culture media prevents homophilic interactions between E-cadherin molecules of the neighboring cells. When corneal epithelial cells were cultured in low Ca2+ medium, E-cadherin interactions were inhibited, but Cx43 gap junctions were not inhibited, confirming that the gap junction assembly did not require E-cadherin interactions between the adjacent cells. When the cells were then provided with Ca2+ in the medium, they formed the E-cadherin-based adherens junctions in the absence of the ROCK inhibitor but not in its presence. However, there was a significant increase in the Cx43 gap junctions when the ROCK activity was inhibited. Thus, downregulation of the Rho/ROCK signaling pathway was found to promote Cx43 gap junction formation. On the basis of this finding we would have expected to see increased Cx43 gap junctions in the limbal epithelium, which lacks ROCK-I activity. Quite the opposite, the limbal epithelium has been reported to be devoid of Cx43 and gap junctions. It is likely that the expression of Cx43 is suppressed in the limbal cells, independently of Rho/ROCK signaling pathways. However, Rho signaling via another downstream target is likely to be involved in promoting Cx43 gap junction assembly because inactivation of Rho was found to inhibit Cx43 gap junction assembly. In addition to ROCK-I and ROCK-II, several other Rho effectors have been identified including PKN/PRK1/PRK2, 57 citron kinase, 58 p140mDia (mDia1), 59 PIP5K, 60 61 myosin phosphatase, 62 and rhophilin, rhotekin, and kinectin. 63 Of these Rho targets, mDia1 has received much attention in recent years. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis. 64 We speculate that mDia1may be involved in regulation of the assembly of Cx43 gap junction based on the findings that mDia1 65 is involved in the formation and orientation of stable microtubules which also interact with Cx43. Tubulin binds the juxtamembrane region of Cx43, which also sediments with tubulin in pull down experiments. 66 If microtubules are involved in the assembly of Cx43 gap junctions, mDia1, which is involved in the formation of microtubules, is likely to play a role in the assembly of the gap junctions. One way to test this speculation would be to find out whether overexpression of constitutively inactive mDia1 results in inhibition of Cx43 gap junction assembly. 
In conclusion, as summarized in Figure 8 , contrary to previously reported findings in other cell types, the present study indicated that E-cadherin adherens junctions are not a prerequisite for gap junction assembly in corneal epithelial cells. Although the Rho/ROCK signaling pathway is involved in the formation of E-cadherin adherens junctions, its downregulation promotes the formation of Cx43 gap junctions. A different Rho signaling pathway than the Rho/ROCK pathway is involved in the assembly of Cx43 gap junctions. A fine balance between the Rho/ROCK pathway and another Rho pathway, involved in gap junction formation, may regulate the number of gap junctions during growth and differentiation of corneal epithelial cells and possibly in other cell types. 
 
Figure 1.
 
The effect of Rho inhibition by C3 exoenzyme on E-cadherin and connexin 43 (Cx43) distribution in corneal epithelial cells in primary cultures. Cells were microinjected with C3 exoenzyme and rhodamine dextran and were analyzed by an indirect immunofluorescence technique using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies as the primary antibodies. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibodies. (A and E) Rhodamine fluorescence of the cells microinjected with rhodamine dextran alone (controls); (B and F) cells microinjected with rhodamine dextran and C3 exoenzyme. (C and D) Immunofluorescence of the cells in the same field as (A) and (B), respectively, stained for E-cadherin. (G and H) Immunofluorescence of the cells in the same field as (E) and (F), respectively, stained for Cx43. Note the loss of E-cadherin-based adherens junctions (D) and reduction in Cx43 gap junctions (H) in C3-injected cells but not in the controls (C) and (G). Bar, 30 μm.
Figure 1.
 
The effect of Rho inhibition by C3 exoenzyme on E-cadherin and connexin 43 (Cx43) distribution in corneal epithelial cells in primary cultures. Cells were microinjected with C3 exoenzyme and rhodamine dextran and were analyzed by an indirect immunofluorescence technique using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies as the primary antibodies. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibodies. (A and E) Rhodamine fluorescence of the cells microinjected with rhodamine dextran alone (controls); (B and F) cells microinjected with rhodamine dextran and C3 exoenzyme. (C and D) Immunofluorescence of the cells in the same field as (A) and (B), respectively, stained for E-cadherin. (G and H) Immunofluorescence of the cells in the same field as (E) and (F), respectively, stained for Cx43. Note the loss of E-cadherin-based adherens junctions (D) and reduction in Cx43 gap junctions (H) in C3-injected cells but not in the controls (C) and (G). Bar, 30 μm.
Figure 2.
 
Effects of Rho kinase (ROCK) inhibition on E-cadherin distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for E-cadherin using a mouse anti-E-cadherin monoclonal antibody and Alexa 488-conjugated goat anti-mouse IgG secondary antibody. The nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note loss of E-cadherin staining at the regions of cell-cell contacts in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 2.
 
Effects of Rho kinase (ROCK) inhibition on E-cadherin distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for E-cadherin using a mouse anti-E-cadherin monoclonal antibody and Alexa 488-conjugated goat anti-mouse IgG secondary antibody. The nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note loss of E-cadherin staining at the regions of cell-cell contacts in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 3.
 
Effects of Rho kinase (ROCK) inhibition on Cx43 distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for Cx43, using rabbit anti-Cx43 polyclonal antibodies and an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody. Nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note an increase in the number of gap junctions and increased Cx43 staining in perinuclear regions in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 3.
 
Effects of Rho kinase (ROCK) inhibition on Cx43 distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for Cx43, using rabbit anti-Cx43 polyclonal antibodies and an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody. Nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note an increase in the number of gap junctions and increased Cx43 staining in perinuclear regions in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 4.
 
Western blot analysis of the changes in the cellular levels of Cx43 upon inactivation of ROCK. Corneal epithelial cells were grown in duplicate sets in culture, and one set was treated with ROCK inhibitor (10 μM) and the other without inhibitor (control). Cells were extracted in Triton X-100-containing buffer, and the soluble (S) and insoluble (I) fractions were analyzed by SDS-PAGE, followed by Western blot analyses. The blot on the left was stained with Coomassie blue, and on the right is the Western blot probed with rabbit anti-Cx43 antibodies.
Figure 4.
 
Western blot analysis of the changes in the cellular levels of Cx43 upon inactivation of ROCK. Corneal epithelial cells were grown in duplicate sets in culture, and one set was treated with ROCK inhibitor (10 μM) and the other without inhibitor (control). Cells were extracted in Triton X-100-containing buffer, and the soluble (S) and insoluble (I) fractions were analyzed by SDS-PAGE, followed by Western blot analyses. The blot on the left was stained with Coomassie blue, and on the right is the Western blot probed with rabbit anti-Cx43 antibodies.
Figure 5.
 
Western blot analysis of the changes in the cellular levels of E-cadherin and gap junction-associated Cx43 upon ROCK inhibition. Identical sets of P1 cultures of rabbit corneal epithelial cells were either treated with Y-27632 or not treated (controls) for 6, 12, and 18 hours. The cells were extracted in a RIPA buffer for the analysis of E-cadherin or with a Triton X-100-containing buffer for the analysis of the insoluble fraction, which contains gap junction-associated Cx43. A total of 20 μg of protein was loaded per lane except the last two lanes (18 hours treatment) in the right panel, which were loaded with 4 μg of protein per lane. Densitometric analysis of the intensities of the bands in the x-ray films were normalized with major protein bands in Coomassie blue-stained blots, and the data in the bar graph are the relative intensities of the immunoreactive bands compared with those in the controls at 6 hours. The arrows in the left panel point to the migration distance of a 120-kDa protein (E-cadherin) and in the right panel point to that of a 43-kDa protein (connexin 43).
Figure 5.
 
Western blot analysis of the changes in the cellular levels of E-cadherin and gap junction-associated Cx43 upon ROCK inhibition. Identical sets of P1 cultures of rabbit corneal epithelial cells were either treated with Y-27632 or not treated (controls) for 6, 12, and 18 hours. The cells were extracted in a RIPA buffer for the analysis of E-cadherin or with a Triton X-100-containing buffer for the analysis of the insoluble fraction, which contains gap junction-associated Cx43. A total of 20 μg of protein was loaded per lane except the last two lanes (18 hours treatment) in the right panel, which were loaded with 4 μg of protein per lane. Densitometric analysis of the intensities of the bands in the x-ray films were normalized with major protein bands in Coomassie blue-stained blots, and the data in the bar graph are the relative intensities of the immunoreactive bands compared with those in the controls at 6 hours. The arrows in the left panel point to the migration distance of a 120-kDa protein (E-cadherin) and in the right panel point to that of a 43-kDa protein (connexin 43).
Figure 6.
 
Analysis of functional gap junctions by Lucifer yellow dye transfer. Lucifer yellow and rhodamine dextran were scrape-loaded by making a scrape line in monolayers of corneal epithelial cells in culture, either control cells (A) or cells pretreated for 18 hours with Y-27632 (B). (C and D) Phase contrast micrographs of (A) and (B), respectively. The cells that are stained red (rhodamine dextran) on either sides of the scrape line are the cells that were originally loaded with Lucifer yellow. Lines drawn in (A) and (B) show the distance of the lateral transfer of Lucifer yellow.
Figure 6.
 
Analysis of functional gap junctions by Lucifer yellow dye transfer. Lucifer yellow and rhodamine dextran were scrape-loaded by making a scrape line in monolayers of corneal epithelial cells in culture, either control cells (A) or cells pretreated for 18 hours with Y-27632 (B). (C and D) Phase contrast micrographs of (A) and (B), respectively. The cells that are stained red (rhodamine dextran) on either sides of the scrape line are the cells that were originally loaded with Lucifer yellow. Lines drawn in (A) and (B) show the distance of the lateral transfer of Lucifer yellow.
Figure 7.
 
Immunofluorescence analyses of the effects of low Ca2+ on E-cadherin adherens junction formation and Cx43 gap junction assembly. Corneal epithelial cells in P0 were cultured in low Ca2+ medium (A and D); the medium was replaced with the medium containing normal levels of Ca2+ with no inhibitor (B and E) and with Y-27632 (C and F) for 4 hours. An indirect immunofluorescence staining of E-cadherin (AC) and Cx43 (DF) using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies, respectively. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibody. Nuclei were stained with propidium iodide. Bar, 20 μm.
Figure 7.
 
Immunofluorescence analyses of the effects of low Ca2+ on E-cadherin adherens junction formation and Cx43 gap junction assembly. Corneal epithelial cells in P0 were cultured in low Ca2+ medium (A and D); the medium was replaced with the medium containing normal levels of Ca2+ with no inhibitor (B and E) and with Y-27632 (C and F) for 4 hours. An indirect immunofluorescence staining of E-cadherin (AC) and Cx43 (DF) using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies, respectively. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibody. Nuclei were stained with propidium iodide. Bar, 20 μm.
Figure 8.
 
A model showing possible regulators of the assembly of E-cadherin adherens junctions and Cx43 gap junctions. Although the Rho/ROCK pathway regulates the assembly of E-cadherin adherens junctions in corneal epithelial cells, its downregulation increases the number of gap junctions. E-cadherin assembly is not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Rac1, Cdc42, and Rho have been shown to be required for E-cadherin-mediated cell-cell adhesion in MDCK cells and keratinicytes. 21 30 32 67 68 Cadherins form tight complexes with catenins, which are thought to link cadherins to actin filaments (for review, see Ref. 69 ). Activation of the wnt-1 signaling pathway has been shown to promote catenin-cadherin complex formation and enhance cadherin-mediated cell-cell adhesion in certain cell types. 70 Signaling pathways involved in the regulation of Cx43 gap junction assembly have not been identified.
Figure 8.
 
A model showing possible regulators of the assembly of E-cadherin adherens junctions and Cx43 gap junctions. Although the Rho/ROCK pathway regulates the assembly of E-cadherin adherens junctions in corneal epithelial cells, its downregulation increases the number of gap junctions. E-cadherin assembly is not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Rac1, Cdc42, and Rho have been shown to be required for E-cadherin-mediated cell-cell adhesion in MDCK cells and keratinicytes. 21 30 32 67 68 Cadherins form tight complexes with catenins, which are thought to link cadherins to actin filaments (for review, see Ref. 69 ). Activation of the wnt-1 signaling pathway has been shown to promote catenin-cadherin complex formation and enhance cadherin-mediated cell-cell adhesion in certain cell types. 70 Signaling pathways involved in the regulation of Cx43 gap junction assembly have not been identified.
The authors thank Welfide Corporation for the kind gift of Y-27632. 
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Figure 1.
 
The effect of Rho inhibition by C3 exoenzyme on E-cadherin and connexin 43 (Cx43) distribution in corneal epithelial cells in primary cultures. Cells were microinjected with C3 exoenzyme and rhodamine dextran and were analyzed by an indirect immunofluorescence technique using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies as the primary antibodies. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibodies. (A and E) Rhodamine fluorescence of the cells microinjected with rhodamine dextran alone (controls); (B and F) cells microinjected with rhodamine dextran and C3 exoenzyme. (C and D) Immunofluorescence of the cells in the same field as (A) and (B), respectively, stained for E-cadherin. (G and H) Immunofluorescence of the cells in the same field as (E) and (F), respectively, stained for Cx43. Note the loss of E-cadherin-based adherens junctions (D) and reduction in Cx43 gap junctions (H) in C3-injected cells but not in the controls (C) and (G). Bar, 30 μm.
Figure 1.
 
The effect of Rho inhibition by C3 exoenzyme on E-cadherin and connexin 43 (Cx43) distribution in corneal epithelial cells in primary cultures. Cells were microinjected with C3 exoenzyme and rhodamine dextran and were analyzed by an indirect immunofluorescence technique using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies as the primary antibodies. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibodies. (A and E) Rhodamine fluorescence of the cells microinjected with rhodamine dextran alone (controls); (B and F) cells microinjected with rhodamine dextran and C3 exoenzyme. (C and D) Immunofluorescence of the cells in the same field as (A) and (B), respectively, stained for E-cadherin. (G and H) Immunofluorescence of the cells in the same field as (E) and (F), respectively, stained for Cx43. Note the loss of E-cadherin-based adherens junctions (D) and reduction in Cx43 gap junctions (H) in C3-injected cells but not in the controls (C) and (G). Bar, 30 μm.
Figure 2.
 
Effects of Rho kinase (ROCK) inhibition on E-cadherin distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for E-cadherin using a mouse anti-E-cadherin monoclonal antibody and Alexa 488-conjugated goat anti-mouse IgG secondary antibody. The nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note loss of E-cadherin staining at the regions of cell-cell contacts in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 2.
 
Effects of Rho kinase (ROCK) inhibition on E-cadherin distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for E-cadherin using a mouse anti-E-cadherin monoclonal antibody and Alexa 488-conjugated goat anti-mouse IgG secondary antibody. The nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note loss of E-cadherin staining at the regions of cell-cell contacts in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 3.
 
Effects of Rho kinase (ROCK) inhibition on Cx43 distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for Cx43, using rabbit anti-Cx43 polyclonal antibodies and an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody. Nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note an increase in the number of gap junctions and increased Cx43 staining in perinuclear regions in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 3.
 
Effects of Rho kinase (ROCK) inhibition on Cx43 distribution in corneal epithelial cells. Corneal epithelial cells in culture, treated with the ROCK inhibitor, Y-27632, for 6 (D), 12 (E), and 18 hours (F) and corresponding nontreated controls (A, B, and C, respectively) were immunostained for Cx43, using rabbit anti-Cx43 polyclonal antibodies and an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody. Nuclei were stained with propidium iodide. Presented here are two color-merged images, derived from projected z-series images of green and red fluorescence, which were collected sequentially at 0.25-μm focus intervals. Note an increase in the number of gap junctions and increased Cx43 staining in perinuclear regions in Y-27632-treated cells (DF). Bar, 30 μm.
Figure 4.
 
Western blot analysis of the changes in the cellular levels of Cx43 upon inactivation of ROCK. Corneal epithelial cells were grown in duplicate sets in culture, and one set was treated with ROCK inhibitor (10 μM) and the other without inhibitor (control). Cells were extracted in Triton X-100-containing buffer, and the soluble (S) and insoluble (I) fractions were analyzed by SDS-PAGE, followed by Western blot analyses. The blot on the left was stained with Coomassie blue, and on the right is the Western blot probed with rabbit anti-Cx43 antibodies.
Figure 4.
 
Western blot analysis of the changes in the cellular levels of Cx43 upon inactivation of ROCK. Corneal epithelial cells were grown in duplicate sets in culture, and one set was treated with ROCK inhibitor (10 μM) and the other without inhibitor (control). Cells were extracted in Triton X-100-containing buffer, and the soluble (S) and insoluble (I) fractions were analyzed by SDS-PAGE, followed by Western blot analyses. The blot on the left was stained with Coomassie blue, and on the right is the Western blot probed with rabbit anti-Cx43 antibodies.
Figure 5.
 
Western blot analysis of the changes in the cellular levels of E-cadherin and gap junction-associated Cx43 upon ROCK inhibition. Identical sets of P1 cultures of rabbit corneal epithelial cells were either treated with Y-27632 or not treated (controls) for 6, 12, and 18 hours. The cells were extracted in a RIPA buffer for the analysis of E-cadherin or with a Triton X-100-containing buffer for the analysis of the insoluble fraction, which contains gap junction-associated Cx43. A total of 20 μg of protein was loaded per lane except the last two lanes (18 hours treatment) in the right panel, which were loaded with 4 μg of protein per lane. Densitometric analysis of the intensities of the bands in the x-ray films were normalized with major protein bands in Coomassie blue-stained blots, and the data in the bar graph are the relative intensities of the immunoreactive bands compared with those in the controls at 6 hours. The arrows in the left panel point to the migration distance of a 120-kDa protein (E-cadherin) and in the right panel point to that of a 43-kDa protein (connexin 43).
Figure 5.
 
Western blot analysis of the changes in the cellular levels of E-cadherin and gap junction-associated Cx43 upon ROCK inhibition. Identical sets of P1 cultures of rabbit corneal epithelial cells were either treated with Y-27632 or not treated (controls) for 6, 12, and 18 hours. The cells were extracted in a RIPA buffer for the analysis of E-cadherin or with a Triton X-100-containing buffer for the analysis of the insoluble fraction, which contains gap junction-associated Cx43. A total of 20 μg of protein was loaded per lane except the last two lanes (18 hours treatment) in the right panel, which were loaded with 4 μg of protein per lane. Densitometric analysis of the intensities of the bands in the x-ray films were normalized with major protein bands in Coomassie blue-stained blots, and the data in the bar graph are the relative intensities of the immunoreactive bands compared with those in the controls at 6 hours. The arrows in the left panel point to the migration distance of a 120-kDa protein (E-cadherin) and in the right panel point to that of a 43-kDa protein (connexin 43).
Figure 6.
 
Analysis of functional gap junctions by Lucifer yellow dye transfer. Lucifer yellow and rhodamine dextran were scrape-loaded by making a scrape line in monolayers of corneal epithelial cells in culture, either control cells (A) or cells pretreated for 18 hours with Y-27632 (B). (C and D) Phase contrast micrographs of (A) and (B), respectively. The cells that are stained red (rhodamine dextran) on either sides of the scrape line are the cells that were originally loaded with Lucifer yellow. Lines drawn in (A) and (B) show the distance of the lateral transfer of Lucifer yellow.
Figure 6.
 
Analysis of functional gap junctions by Lucifer yellow dye transfer. Lucifer yellow and rhodamine dextran were scrape-loaded by making a scrape line in monolayers of corneal epithelial cells in culture, either control cells (A) or cells pretreated for 18 hours with Y-27632 (B). (C and D) Phase contrast micrographs of (A) and (B), respectively. The cells that are stained red (rhodamine dextran) on either sides of the scrape line are the cells that were originally loaded with Lucifer yellow. Lines drawn in (A) and (B) show the distance of the lateral transfer of Lucifer yellow.
Figure 7.
 
Immunofluorescence analyses of the effects of low Ca2+ on E-cadherin adherens junction formation and Cx43 gap junction assembly. Corneal epithelial cells in P0 were cultured in low Ca2+ medium (A and D); the medium was replaced with the medium containing normal levels of Ca2+ with no inhibitor (B and E) and with Y-27632 (C and F) for 4 hours. An indirect immunofluorescence staining of E-cadherin (AC) and Cx43 (DF) using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies, respectively. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibody. Nuclei were stained with propidium iodide. Bar, 20 μm.
Figure 7.
 
Immunofluorescence analyses of the effects of low Ca2+ on E-cadherin adherens junction formation and Cx43 gap junction assembly. Corneal epithelial cells in P0 were cultured in low Ca2+ medium (A and D); the medium was replaced with the medium containing normal levels of Ca2+ with no inhibitor (B and E) and with Y-27632 (C and F) for 4 hours. An indirect immunofluorescence staining of E-cadherin (AC) and Cx43 (DF) using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies, respectively. Alexa 488-conjugated goat anti-mouse IgG and anti-rabbit IgG, respectively, were used as the secondary antibody. Nuclei were stained with propidium iodide. Bar, 20 μm.
Figure 8.
 
A model showing possible regulators of the assembly of E-cadherin adherens junctions and Cx43 gap junctions. Although the Rho/ROCK pathway regulates the assembly of E-cadherin adherens junctions in corneal epithelial cells, its downregulation increases the number of gap junctions. E-cadherin assembly is not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Rac1, Cdc42, and Rho have been shown to be required for E-cadherin-mediated cell-cell adhesion in MDCK cells and keratinicytes. 21 30 32 67 68 Cadherins form tight complexes with catenins, which are thought to link cadherins to actin filaments (for review, see Ref. 69 ). Activation of the wnt-1 signaling pathway has been shown to promote catenin-cadherin complex formation and enhance cadherin-mediated cell-cell adhesion in certain cell types. 70 Signaling pathways involved in the regulation of Cx43 gap junction assembly have not been identified.
Figure 8.
 
A model showing possible regulators of the assembly of E-cadherin adherens junctions and Cx43 gap junctions. Although the Rho/ROCK pathway regulates the assembly of E-cadherin adherens junctions in corneal epithelial cells, its downregulation increases the number of gap junctions. E-cadherin assembly is not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Rac1, Cdc42, and Rho have been shown to be required for E-cadherin-mediated cell-cell adhesion in MDCK cells and keratinicytes. 21 30 32 67 68 Cadherins form tight complexes with catenins, which are thought to link cadherins to actin filaments (for review, see Ref. 69 ). Activation of the wnt-1 signaling pathway has been shown to promote catenin-cadherin complex formation and enhance cadherin-mediated cell-cell adhesion in certain cell types. 70 Signaling pathways involved in the regulation of Cx43 gap junction assembly have not been identified.
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