title: 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.
abstract: In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation 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-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Y h
authors.initials: Yh
authors.lastname: Chen
authors.forename: Q
authors.initials: Q
authors.lastname: Lu
authors.forename: E E
authors.initials: EE
authors.lastname: Schneeberger
authors.forename: D A
authors.initials: DA
authors.lastname: Goodenough
mesh_terms: D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology; D004285:Dogs; D015536:Down-Regulation; D004848:Epithelium / Q000502:physiology; D006207:Half-Life; D007668:Kidney / Q000166:cytology; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D019108:Tight Junctions / Q000737:chemistry* / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D018631:ras Proteins / Q000502:physiology*
chemical_list: D015820:Cadherins; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D014443:Tyrosine; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins