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 cad- herins 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- Investigative Ophthalmology & Visual Science, April 2002, Vol. 43, No. 4 978 Copyright © Association for Research in Vision and Ophthalmology Downloaded from jov.arvojournals.org on 09/26/2021 IOVS, April 2002, Vol. 43, No. 4 Rho Signaling in Adherens and Gap Junctions 979 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