Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium

Invest Ophthalmol Vis Sci. 2002 Apr;43(4):978-86.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adherens Junctions / metabolism*
  • Animals
  • Blotting, Western
  • Cadherins / metabolism
  • Cell Adhesion
  • Cells, Cultured
  • Connexin 43 / metabolism
  • Down-Regulation
  • Enzyme Inhibitors / pharmacology
  • Epithelium, Corneal / cytology
  • Epithelium, Corneal / metabolism*
  • Fluorescent Antibody Technique, Indirect
  • Gap Junctions / metabolism*
  • Intracellular Signaling Peptides and Proteins
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / metabolism*
  • Rabbits
  • Signal Transduction / physiology*
  • rho GTP-Binding Proteins / antagonists & inhibitors
  • rho GTP-Binding Proteins / metabolism*
  • rho-Associated Kinases

Substances

  • Cadherins
  • Connexin 43
  • Enzyme Inhibitors
  • Intracellular Signaling Peptides and Proteins
  • Protein Serine-Threonine Kinases
  • rho-Associated Kinases
  • rho GTP-Binding Proteins