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Vol. 11, Issue 3, 849-862, March 2000
and
*Department of Cell Biology, Harvard Medical School, Boston,
Massachusetts 02115; 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.
Center for Neurologic Diseases,
Brigham and Women's Hospital, and Department of Neurology, Harvard
Medical School, Boston, Massachusetts 02115; and
Department of Pathology, Massachusetts General Hospital,
Boston, Massachusetts 02114
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