Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in Ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludi n and ZO-1 are structural components of the tight junctions that seal the p aracellular spaces between the cells and contribute to the epithelial barri er function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-l, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly exp ressed. After treatment of the Ras-transformed cells with the mitogen-activ ated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activ ation of mitogen-activated protein kinase (MAPK), occludin, claudin-l, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, a nd E-cadherin protein expression was induced. Although it is generally beli eved that E-cadherin-mediated cell-cell adhesion is required for tight junc tion assembly, the recruitment of occludin to the cell-cell contact area an d the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold i ncrease in the transepithelial electrical resistance indicated the formatio n of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO -1, but not claudin-l, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of ep ithelial cell morphology and the assembly of tight junctions in Pas-transfo rmed epithelial cells and that tyrosine phosphorylation of occludin and ZO- 1 may play a role in some aspects of tight junction formation.