CYTOSKELETAL INVOLVEMENT IN THE MODULATION OF CELL-CELL JUNCTIONS BY THE PROTEIN-KINASE INHIBITOR H-7

The protein kinase inhibitor H-7 has been shown to block junction diss ociation induced by low extracellular calcium in Madin Darby canine ki dney epithelial cells (S. Citi, J. Cell Biol. (1992) 117, 169-178). To understand the basis of this effect, we have examined how H-7 affects the organization of junctions and the actin cytoskeleton in different types of epithelial cells in culture. Immunofluorescence microscopy s howed that H-7 confers Ca2+ independence on cultured epithelial lens c ells, which lack tight junctions and desmosomes but have microfilament -associated adherens junctions. In these cells, H-7 did not protect N- cadherin from trypsin digestion at low extracellular calcium, suggesti ng that H-7 does not stabilize the 'active' cadherin conformation. In cultured Madin Darby canine kidney cells, H-7 partially prevented the fall in transepithelial resistance induced by cytochalasin D, either a lone or in conjunction with calcium chelators. Double-immunofluorescen ce microscopy showed that H-7 inhibits both the fragmentation of label ing for the tight junction protein cingulin and the condensation of ac tin into cytoplasmic foci induced by cytochalasin D. Taken together, t hese observations indicate that H-7 inhibits junction dissociation by affecting the contractility of the adherens junction-associated microf ilaments following treatment with calcium chelators or cytochalasin D.