Signaling in Adherens and Gap Junction Assembly in Corneal Epithelium

Rho and Rho-Kinase (ROCK) Signaling in Adherens and Gap Junction Assembly in Corneal Epithelium

Susan C. Anderson, Cynthia Stone, Lisa Tkach, and Nirmala SundarRaj

PURPOSE. To examine whether Rho and its downstream target, tion of the transmembrane connexon from one cell with the a Rho-associated kinase (ROCK), are involved in the regulation connexon of the adjoining cell. The turnover time of Cx has of the assembly of cadherin-mediated cell adhesion and con- been reported to be very short, with a half-life of only 1.3–2 nexin 43 (Cx43) gap junctions in corneal epithelium. hours.6–8 The mechanisms of the transport of connexins to the METHODS. Rho and ROCK activities in rabbit corneal epithelial cell membranes from the Golgi and the alignment of opposing cells in culture were inhibited by microinjection of a Clostrid- connexons to form the gap junction channels are poorly un- ium botulinum ADP-ribosyltransferase (C3) and treatment derstood, and the possible interacting molecules involved in with a ROCK specific inhibitor (Y-27632), respectively. Immu- the assembly of gap junctions have not yet been identified. nocytochemical and Western blot techniques were used to Several reported findings suggest that the formation of cad- study the distribution and relative concentrations of E-cadherin herin-based adherens junctions precedes and facilitates the and Cx43. Intercellular communication via gap junctions was assembly of connexons at the cell–cell contact regions.9–14 For measured by a dye transfer assay. example, the inhibition of E-cadherin– or N-cadherin–based cell–cell adhesion, using antibodies to these proteins, has been RESULTS. Inhibition of Rho activity in the primary cultures of 9 rabbit corneal epithelial cells by microinjecting them with C3 shown to inhibit the assembly of gap junctions. In poorly coupled cells, the expression of recombinant cadherins was resulted in an inhibition of the assembly of E-cadherin–based 11 cell–cell adhesion and Cx43 gap junctions. However, inhibi- shown to greatly increase coupling. The expression of cad- tion of the ROCK activity by treatment with Y-27632 inhibited herin did not affect the synthesis of Cxs but increased the phosphorylation of Cx and Cx gap junctions at the cell–cell the assembly of E-cadherin–based cell–cell adhesions but not 12 Cx43 gap junctions. In fact, inhibition of ROCK resulted in an contacts. Immunoelectron microscopic analyses showed a colocalization of connexin, E-cadherin, and ␤-catenin at the increase in the number of Cx43 gap junctions and in cell–cell 15 communication. Culturing corneal epithelial cells in a low cell–cell contact sites during gap junction formation. A more recent report suggests that the effect of cadherins on gap calcium medium prevented the formation of E-cadherin adhe- 16 rens junctions but not the Cx43 gap junctions. junction assembly is cell-type specific. Although an increase in cadherin-based adhesion resulted in an increase in the gap ONCLUSIONS E-cadherin adherens junctions are not a prereq- C . junction communication in hepatoma cells in culture, it has an uisite for the assembly of Cx43 gap junctions in corneal epi- opposite effect in the L cells. E-cadherin has been shown to be thelial cells. Different Rho signaling pathways are involved in involved in controlling the specificity of gap junction forma- the regulation of the assembly of E-cadherin mediated cell–cell tion. When rat epithelial cells expressing P- and 125-kDa N- adhesion and Cx43 gap junctions. Although a Rho/ROCK sig- cadherin are grown in a mixed culture with rat fibroblasts naling pathway influences the assembly of E-cadherin adherens expressing 140-kDa N-cadherin, each cell type established ho- junctions, its downregulation influences Cx43 gap junction mologous communication via Cx43 gap junctions and very assembly. (Invest Ophthalmol Vis Sci. 2002;43:978–986) little heterologous communication. However, transfection of both these cells with E-cadherin resulted in a 10-fold increase ells, in most tissues, communicate through gap junctions 17 Cthat are intercellular hydrophilic channels that allow the in heterologous communication. transfer of cytoplasmic molecules of Ͻ1 kDa between neigh- Cadherin-mediated cell–cell adhesion is formed by the ho- boring cells.1–3 Gap junctions have been implicated to be mophilic interaction of the extracellular domains of the cadherins of the adjacent cells and the interaction of the cytoplas- important in the control of cell proliferation, differentiation, ␤ ␥ and regeneration.4,5 They are composed of connexins (Cxs), a mic domain with catenins ( -catenin, -catenin/plakoglobin, 2 and ␣-catenin) that link E-cadherin to the actin cytoskele- family of related transmembrane proteins. After their synthe- 18–20 sis in the endoplasmic reticulum, Cxs oligomerize into hexam- ton. The Rho family of small GTPases, including Rho, Rac, and Cdc42, have been implicated in the development and eric hemichannels (connexons) in the Golgi and are trans- 21–25 ported to the cell surface. Gap junction channels, connecting maintenance of E-cadherin–mediated cell–cell adhesions. the cytoplasm of the adjoining cells, are formed by an interac- 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.26–29 In MDCK cells, inhibition of RhoA, Rac, or Cdc42 activity has been found to From the Department of Ophthalmology, University of Pittsburgh result in the loss of E-cadherin–mediated cell–cell adhe- School of Medicine, Pittsburgh, Pennsylvania. 30–32 Supported by Eye and Ear Foundation (Pittsburgh, Pennsylvania), sion. In keratinocytes, the inhibition of Rac or RhoA 2ϩ Research to Prevent Blindness, and National Institutes of Health Grants activity has been shown to inhibit the induction of Ca - EYO3263 and Core Grant EYO8098. dependent E-cadherin–mediated cell–cell adhesion.25,33 Based Submitted for publication August 29, 2001; revised November 26, on the current information, it is not clear whether the involve- 2001; accepted December 6, 2001. ment of specific Rho family members in the adherens interac- Commercial relationships policy: N. tions, mediated by specific cadherins, is cell-type specific. The The publication costs of this article were defrayed in part by page knowledge of the downstream events leading to adherens charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. junction formation, which may be a prerequisite for gap junc- Corresponding author: Nirmala SundarRaj, Department of Oph- tion formation, is also limited. Whether the requirement of thalmology, Eye and Ear Institute, 203 Lothrop Street, Pittsburgh, PA adherens interactions in gap junction formation is tissue-type 15213-2588; [email protected]. dependent has not been investigated. If the gap junction for-

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mation were dependent on cadherin-based adherens junction a buffer containing 0.2% Triton X-100 according to McLean and Na- formation, which in turn was regulated by one or more mem- kane.44 The fixed cells were reacted with either 1.5% (for Cx43 bers of the Rho family, then these GTPases should also regulate staining) or 10% (for E-cadherin staining) heat-inactivated goat serum in the gap junction assembly. Although the corneal epithelium phosphate-buffered saline, pH 7.5 (PBS), for 45 minutes to block the has Cx43 gap junctions,34,35 the surrounding limbal epithe- nonspecific binding of the secondary antibody, rinsed with PBS, and lium, which harbors the stem cells of corneal epithelium, lacks then treated with the primary and secondary antibodies using a previ- Cx43 gap junctions.36 Coincidently, an isoform of Rho-associ- ously described technique.45 The primary antibodies included rabbit ated kinase, ROCK-I, is also absent in the limbal epithelium. anti-Cx43 (Zymed Laboratory, Inc., San Francisco, CA) at 1:100 con- The study reported here evaluated whether the Rho signaling centration and mouse monoclonal anti–E-cadherin (BD Transduction cascades, specifically the signaling pathway(s) involving Laboratories, Lexington, KY) at 5 ␮g/mL concentration, and the sec- ROCK,37–40 were involved in the formation of E-cadherin ad- ondary antibodies were Alexa 488–conjugated goat anti-rabbit or anti- herens junctions and whether E-cadherin adherens junction mouse IgG (Molecular Probes), at 1:1500 or 1:2500 concentration, formation was a prerequisite for Cx43 gap junction assembly in respectively. For nuclear staining, the immunostained cells were corneal epithelial cells. treated with 5 ␮g/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 MATERIALS AND METHODS Z-stack images (0.25-␮m interval) were collected sequentially using a Bio-Rad Radiance 2000 (Hertfordshire, UK) confocal scanning laser Cell Culture and Treatments system attached to an Olympus IX70 inverted microscope (Tokyo, All procedures involving rabbits were performed in compliance with Japan). the ARVO Statement for the Use of Animals in Ophthalmic and Vision To visualize the organization of the actin filaments, the cells were Research. Corneas with the adjacent limbus were excised from rabbit fixed and permeabilized as above and incubated with 1:500 dilution of eyes (Pel-Freez Biologicals, Rogers, AK) and used for growing the Texas Red-X–conjugated phalloidin (Molecular Probes) for 45 minutes,

primary cultures (P0), in SHEM (supplemental hormonal epithelial washed with PBS, and mounted as above. 41 42 medium ) according to Ebato et al. Cells in P0 were subcultured, using 0.25% trypsin/EDTA (GIBCO-BRL, Grand Island, NY), into 60-mm Western Blot Analyses tissue culture dishes or four-well chamber tissue culture slides (Nalge- Cultures grown in 60-mm dishes and treated as described earlier were Nunc International, Napersville, IL) at a density of 3 ϫ 104 cells/cm2 extracted in a buffer containing 9.1 mM dibasic sodium phosphate, 1.7 and incubated further to allow them to reach a desired density. mM monobasic sodium phosphate, 150 mM NaCl, pH 7.4, 1% Triton

When identical sets of P1 cells grown in either 35- or 60-mm tissue X-100, 0.03 TIU/mL aprotinin (Sigma Chemical Co., St. Louis, MO), 1 culture dishes or in chamber slides had reached confluency, they were mM sodium orthovanadate, and 100 ␮g/mL phenylmethylsulfonyl flu- treated with the 10 ␮M ROCK inhibitor, Y-27632 (Welfide Pharmaceu- oride (PMSF), and the insoluble fraction was used for the analysis of tical Industries, Osaka, Japan), in SHEM medium or with SHEM without Cx43 in the gap junctions. For the analyses of E-cadherin, the cells the inhibitor (control). After a further incubation of cells in these were extracted in a modified RIPA buffer (50 mM Tris-HCl, 150 mM media for specific lengths of time, the cells were processed for either NaCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, immunostaining or for Western blot analyses. Media with or without 0.03 TIU/mL aprotinin [Sigma], 1 mM sodium orthovanadate, 2 mM inhibitor were changed every 6 hours during the treatments. PMSF, 1 ␮g mL leupeptin, 1 ␮g/mL pepstatin A, and 5 mM EDTA). 2ϩ To grow the cells in low calcium (Ca ) medium, P0 cells growing Briefly, the cells in the dishes were rinsed with cold PBS and then 0.2 in SHEM with normal Ca2ϩ concentration (1.8 mM) were subcultured mL of RIPA buffer was added per dish to lyse the cells. The cell lysate into low Ca2ϩ (5 ␮M) medium (DMEM/HAM F12-deficient medium was scraped and collected, and the dishes were rinsed with an addi- with 2.5 mM glutamine, 0.45 mM leucine, 0.5 mM lysine, 0.14 mM tional 0.1 mL RIPA buffer, which was mixed with the first lysate. The ␮ 2ϩ MgCl2, 0.2 mM MgSO4 and 5 MCa chloride), 10% dialyzed fetal lysate was then passed through a 21-gauge needle and then centrifuged bovine serum and penicillin/streptomycin. After the cells had reached at 10,000g for 20 minutes at 4°C. The proteins in the supernatants confluency, one set of cultures was processed for immunofluorescence were estimated using BCA protein assay reagent (Pierce, Rockford, IL). and Western blot analyses and in the remaining dishes, culture medium Aliquots of samples containing 20 ␮g of proteins were subjected to was replaced with SHEM with or without 10 ␮M Y-2763243 and normal 12% or 7% SDS-PAGE for Cx43 and E-cadherin, respectively. The levels of Ca2ϩ (controls). After an additional 4 hours of incubation, the proteins separated on the SDS-PAGE were electrophoretically trans- cells were processed as above. ferred to a nitrocellulose membrane (Schleicher and Schull, Keene, NH), and after treating the membranes with BLOTTO46 to block the Microinjections nonspecific binding sites, the blots were immunoreacted with anti- Cx43 (1:250) and anti–E-cadherin (1:250) antibodies and the horse- Microinjections were carried out using freshly pulled needles, a Leitz radish peroxidase–conjugated secondary antibodies as described micromanipulator (Deerfield, IL), and a Narashige microinjector (East previously.45 The immunoreactive bands were detected using chemi- Meadow, NY) set at a continuous positive injection pressure of 5 to 7 luminescence reagents (Super Signal West Femto reagent from Pierce), psi. C3 (Clostridium botulinum ADP-ribosyltransferase; Cytoskeleton following the manufacturer’s protocols. The chemiluminescent Inc., Denver, CO) at a concentration of 0.1 mg/mL with 0.4 mg/mL of bands detected on the x-ray film were scanned, and the relative rhodamine-dextran, M 10 ϫ 103 (Molecular Probes, Eugene, OR), in a r differences in their intensities were estimated using the Image-Pro buffer containing 20 mM Tris-HCl, 20 mM NaCl, 1 mM MgCl , 0.1 mM 2 Plus analyses software (Media Cybernetics, Silver Spring, MD) and EDTA, 5 mM 2-mercaptoethanol, pH 7.4, was injected into the cyto- were normalized with intensities of three major protein bands in plasm of the corneal epithelial cells in primary explant cultures, grown duplicate blots stained with Coomassie blue. Data were collected in 60-mm tissue culture dishes. After 4 or 6 hours of incubation, the from a minimum of three different sets of experiments and were cells were fixed and immunoreacted with anti-Cx43 or anti–E-cadherin represented as mean Ϯ SD. The t-test was used to determine antibodies as described below. whether the differences in the relative intensities of the reactive bands were statistically significant. Immunostaining Cultures grown in chamber slides or tissue culture dishes and treated Dye Diffusion as above were rinsed three times with PBS, fixed with 2% paraformal- Gap junction permeability was determined using a Lucifer yellow dehyde-lysine-periodate fixative for 5 minutes, and permeabilized with scrapeloading technique.47 Identical sets of confluent corneal epithe-

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lial 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 ϫ 3 0.05% Lucifer yellow and 1 mg/mL rhodamine dextran, Mr 10 10 (Molecular Probes) in PBS, and a scrape line was made in the mono- layer 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 FIGURE 1. The effect of Rho inhibition by C3 exoenzyme on E-cadherin and connexin 43 (Cx43) distribution in corneal epithelial cells in To analyze whether the Rho signaling pathway(s), involving primary cultures. Cells were microinjected with C3 exoenzyme and ROCKs (ROCK-I and/or ROCK-II), participates in the formation rhodamine dextran and were analyzed by an indirect immunofluores- of E-cadherin adherens junctions and Cx43 gap junctions in cence technique using anti-mouse E-cadherin monoclonal antibody and rabbit anti-Cx43 polyclonal antibodies as the primary antibodies. corneal epithelial cells, the effects of the inhibition of ROCK Alexa 488–conjugated goat anti-mouse IgG and anti-rabbit IgG, respec- with a specific inhibitor, Y-27632, was analyzed in P1 cultures tively, were used as the secondary antibodies. (A and E) Rhodamine of corneal epithelium. To ensure that ROCK was inactivated in fluorescence of the cells microinjected with rhodamine dextran alone the cells treated with Y-27632, a set of cells was analyzed for (controls); (B and F) cells microinjected with rhodamine dextran and the changes in the actin filaments. Within 6 hours of treatment, C3 exoenzyme. (C and D) Immunofluorescence of the cells in the same actin filaments were disrupted in the Y-27632–treated cells, as field as (A) and (B), respectively, stained for E-cadherin. (G and H) evident from the staining of the cells with Texas Red-X–con- Immunofluorescence of the cells in the same field as (E) and (F), respectively, stained for Cx43. Note the loss of E-cadherin–based jugated phalloidin (not shown). These cells also developed adherens junctions (D) and reduction in Cx43 gap junctions (H) in many long cytoplasmic extensions. These changes remained C3-injected cells but not in the controls (C) and (G). Bar, 30 ␮m. 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 evi- Western Blot Analyses of the Changes in dent from the absence of immunostaining, and remained un- E-cadherin and Cx43 in Corneal Epithelial Cells detectable at the cell–cell contacts after 12 and 18 hours of the A comparative Western blot analysis of the cell extracts of the inhibitor treatment (Fig. 2E and 2F, respectively). However, Y-27632–treated and nontreated cells was performed to deter- cells treated with Y-27632 exhibited cytoplasmic immunostain- mine the relative levels of E-cadherin and Cx43. Cx43, assem- ing of E-cadherin. E-cadherin was probably internalized upon bled into gap junctions, has been reported to be insoluble in inactivation of ROCK in these cells. Cx43 staining was not lost Triton X-100.6 The Western blot analyses of the Triton X-100– at the cell–cell contacts after 6 hours of the inhibitor treatment soluble and –insoluble extracts indicated that most of the Cx43 (Fig. 3D), and after 12 and 18 hours there appeared to be an in the cells, treated or not treated with the inhibitor for 18 increase in the Cx43 gap junctions at the lateral surface of the hours, was insoluble in Triton X-100 (Fig. 4). The concentra- cells (Fig. 3E and 3F, respectively). In addition, significant tions of Cx43 in the inhibitor-treated cells were also 1.6 Ϯ perinuclear staining, possibly in the Golgi region, and punctate 0.252 higher than the nontreated cells. Although the levels of gap junction-like intracellular staining was detected in many of E-cadherin in the control and the cells treated with Y-27632 for the cells treated with Y-27632 (Fig. 3, D–F). 6, 12, and 18 hours were not significantly different (Fig. 5, top

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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 Al- exa 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 ﬂuores- cence, which were collected sequentially at 0.25-␮m focus intervals. Note loss of E-cadherin staining at the regions of cell–cell contacts in Y-27632–treated cells (D–F). Bar, 30 ␮m.

left), the concentration of Cx43 increased upon inactivation of number of gap junctions, based on the immunocytochemical ROCK with Y-27632 (Fig. 5, top right). From 6 to 18 hours, as and Western blot analyses. To study whether there was a the cell density increased, the concentration of Cx43 also change in intercellular communication, the extent of cell–cell increased to varying extents (8–10-fold) in different experi- diffusion of Lucifer yellow in the cells treated with Y-27632 for ments. However, the concentrations of Cx43 were always 18 hours was compared with control nontreated cells. Figure 6 higher in the Y-27632–treated cells compared with corre- shows typical results. There was a 1.36 Ϯ 0.08-fold increase in sponding concentrations in the nontreated controls. The bar the distance of the dye diffusion in the cells treated with the graphs in Figure 5 (bottom) show the relative differences in the inhibitor. In conﬂuent monolayers of corneal epithelial cells, intensities of immunoreactive bands in the Western blot anal- the depth of lateral dye diffusion was 9 or 10 cells in the ysis of one representative experiment. The intensities of im- controls and 13 or 14 cells in the ROCK-inhibited cells. munoreactive bands in the inhibitor-treated cells, as determined from three different experiments, were 1.5 Ϯ 0.2-, Effects of Inhibition of E-cadherin Junctions on 1.53 Ϯ 0.1-, and 1.6 Ϯ 0.14-fold higher than the controls at 6, Cx43 Gap Junction Formation Ͻ Ͻ Ͻ 12, and 18 hours, respectively (P 0.001, P 0.001, and P The formation of E-cadherin adherens junctions is inhibited in 0.001, respectively). keratinocytes21 by reducing the concentration of Ca2ϩ in the

culture medium. A similar effect was also evident in P1 corneal Effects of ROCK Inhibition on ϩ epithelial cells grown in low Ca2 medium (Fig. 7A). However, Intercellular Communication Cx43 gap junctions were present in low Ca2ϩ medium (Fig. Inhibition of ROCK activity in the corneal epithelial cells re- 7D). In another set of cultures, the low Ca2ϩ medium was sulted in an increase of the concentration of Cx43 and the replaced with medium containing the normal Ca2ϩ concentra-

FIGURE 3. Effects of Rho kinase (ROCK) inhibition on Cx43 distribution in corneal epithelial cells. Cor- neal 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. Pre- sented here are two color-merged images, derived from projected z-- series images of green and red ﬂuo- rescence, which were collected sequentially at 0.25-␮m focus intervals. Note an increase in the number of gap junctions and increased Cx43 staining in perinuclear regions in Y-27632–treated cells (D–F). Bar, 30 ␮m.

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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 ␣3␤1 integrin promotes gap junction assembly, and this process requires Rho signaling. Rho signaling is involved in the formation of stress fibers and focal adhesions27–29 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 (collec- tively 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 FIGURE 4. Western blot analysis of the changes in the cellular levels of specific inhibitor of the ROCK family. When ROCK was inhib- Cx43 upon inactivation of ROCK. Corneal epithelial cells were grown in duplicate sets in culture, and one set was treated with ROCK ited by treating corneal epithelial cells with Y-27632, it dis- inhibitor (10 ␮M) and the other without inhibitor (control). Cells were rupted actin stress fibers in corneal epithelial cells, as ex- extracted in Triton X-100–containing buffer, and the soluble (S) and pected. Based on the immunofluorescence analysis, ROCK insoluble (I) fractions were analyzed by SDS-PAGE, followed by West- inhibition resulted in the elimination of E-cadherin–based ad- ern blot analyses. The blot on the left was stained with Coomassie blue, herens junctions at the cell–cell contacts. However, it did not and on the right is the Western blot probed with rabbit anti-Cx43 inhibit the assembly of Cx43 gap junctions. In fact, inhibition antibodies. 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 tion in the presence or absence (control) of inhibitor, Y-27632. junctions as evident from the dye diffusion study. We currently After 4 hours of further incubation, although E-cadherin junc- do not know whether other gap junctions, such as connexin tions were assembled and were evident at the lateral surfaces 50 gap junctions, also accounted for an increase in functional in the controls (Fig. 7B), they were absent in the cultures gap junctions in ROCK-inhibited cells. Although E-cadherin treated with Y-27632 (Fig. 7C). Cx43 junctions were evident in was diminished from the cell–cell contacts in Y-27632–treated 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.9–14 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 Cdc42.22–25 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 cells48 as well as corneal epithelial cells49 express E-cadherin. Gap junctions composed of Cx43 and Cx50 have been identified in corneal 34–36 FIGURE 5. Western blot analysis of the changes in the cellular levels of epithelium. E-cadherin and gap junction–associated Cx43 upon ROCK inhibition. In the present study, the possible involvement of Rho sig- Identical sets of P1 cultures of rabbit corneal epithelial cells were either naling pathway(s), in the formation of E-cadherin–based adhe- treated with Y-27632 or not treated (controls) for 6, 12, and 18 hours. rens junctions and the assembly of Cx43 gap junctions were The cells were extracted in a RIPA buffer for the analysis of E-cadherin evaluated by studying the consequence of Rho inhibition in or with a Triton X-100–containing buffer for the analysis of the insol- corneal epithelial cells. When Rho activity was inhibited in the uble fraction, which contains gap junction-associated Cx43. A total of primary cultures of rabbit corneal epithelial cells by injecting 20 ␮g of protein was loaded per lane except the last two lanes (18 C3, E-cadherin adherens junctions were diminished at cell–cell hours treatment) in the right panel, which were loaded with 4 ␮gof contact regions, indicating that Rho signaling pathway(s) was protein per lane. Densitometric analysis of the intensities of the bands in the x-ray films were normalized with major protein bands in Coo- involved in their assembly. Rho involvement in the assembly of massie blue–stained blots, and the data in the bar graph are the E-cadherin–based adherens junctions in corneal epithelium relative intensities of the immunoreactive bands compared with those conformed with the previously reported findings in the MDCK in the controls at 6 hours. The arrows in the left panel point to the 30–32 50 cell line and keratinocytes. It was interesting to note migration distance of a 120-kDa protein (E-cadherin) and in the right that Cx43 gap junctions were also lost in the cells injected with panel point to that of a 43-kDa protein (connexin 43).

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FIGURE 6. Analysis of functional gap junctions by Lucifer yellow dye transfer. Lucifer yellow and rhodamine dextran were scrape-loaded by mak- ing a scrape line in monolayers of corneal epithelial cells in culture, either control cells (A) or cells pre- treated for 18 hours with Y-27632 (B). (C and D) Phase contrast micro- graphs 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 Lu- cifer yellow.

cells, it was detectable in the cytoplasm of the cells. The total tected even after 18 hours of inhibitor treatment. E-cadherin is levels of E-cadherin in the inhibitor-treated cells were not a transmembrane protein, and its adhesive function is brought signiﬁcantly different from the controls. Thus, ROCK-I signal- about by Ca2ϩ-dependent homophilic interaction between E- ing most likely participates in the actin ﬁlament–mediated cadherin molecules of the adjacent cells. The cytoplasmic tail translocation of E-cadherin to the cell membrane. Contrary to of E-cadherin interacts with the actin cytoskeleton via ␤-cate- the present observation, the inhibition of ROCK with Y-27632 nin, and this complex formation aides in the clustering of the did not prevent clustering of cadherin during induction of receptors and strengthening of cell–cell adhesion.18–20 The cell–cell adhesion in keratinocytes in culture.33 However, interaction of the cytoskeleton not only is involved in the blocking of ROCK function by using a dominant negative clustering of the E-cadherin receptors but also in providing a approach was reported to partially perturb the localization of framework for the different cytoskeletal proteins and signaling cadherin receptors in MDCK cells.55 In the absence of E- molecules at cell–cell junctions.56 The lowering of the Ca2ϩ cadherin adherens junctions, Cx43 gap junctions were de- concentration in tissue culture media prevents homophilic

FIGURE 7. Immunoﬂuorescence analyses of the effects of low Ca2ϩ on E- cadherin adherens junction formation and Cx43 gap junction assembly. Cor-

neal epithelial cells in P0 were cultured in low Ca2ϩ medium (A and D); the medium was replaced with the medium containing normal levels of Ca2ϩ with no inhibitor (B and E) and with Y-27632 (C and F) for 4 hours. An indirect immunoﬂuorescence staining of E-cadherin (A–C) and Cx43 (D–F) 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 ␮m.

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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 junc- FIGURE 8. A model showing possible regulators of the assembly of tions during growth and differentiation of corneal epithelial E-cadherin adherens junctions and Cx43 gap junctions. Although the cells and possibly in other cell types. 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, Acknowledgment Cdc42, and Rho have been shown to be required for E-cadherin– mediated cell–cell adhesion in MDCK cells and keratini- The authors thank Welfide Corporation for the kind gift of Y-27632. cytes.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 References promote catenin–cadherin complex formation and enhance cadherin- mediated cell–cell adhesion in certain cell types.70 Signaling pathways 1. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and involved in the regulation of Cx43 gap junction assembly have not intercellular communication. 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