Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.
Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.
Macek
R
R
Roche Diagnostics GmbH, Pharma Research Oncology, Dept. Cell Biology, Nonnenwald 2,82377 Penzberg, Germany. robert.macek@roche.com
Swisshelm
K
K
Kubbies
M
M
D001943:Breast Neoplasms / Q000378:metabolism*; D062445:Claudin-1; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D018631:ras Proteins / Q000378:metabolism*
C568108:CLDN1 protein, human; D062445:Claudin-1; D008565:Membrane Proteins; D018631:ras Proteins