Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium.
OBJECTIVE
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.
Anderson
Susan C
SC
Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Stone
Cynthia
C
Tkach
Lisa
L
SundarRaj
Nirmala
N
D022005:Adherens Junctions / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054460:rho-Associated Kinases
D015820:Cadherins; D018031:Connexin 43; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins